Overnight grown primary culture of E. coli cells (1 % v/v final concentration) was inoculated into 1 litre of LB media containing antibiotics. Culture was incubated at 37 oc at 200 rpm. Growth was monitored by measuring absorbance of E. coli broth at 600 nm. Culture was induced by adding 1 mM IPTG at an OD of 0.6 and was harvested after 4 hrs of induction. Samples were taken on an hourly basis after induction to check the kinetics of protein expression. Un-induced and induced E. coli cells were analyzed by SDS-PAGE to check the expression of recombinant protein.

Primary culture of E. coli was grown in LB medium containing either ampicillin (Amp) and/or kanamycin (Kan) to final concentration of 100 j..tg/ml and 25 J..tg/ml respectively. Depending on the vector construct, antibiotics were used for expression of different proteins as described in Table 3.1. Medium was inoculated with 1 ml glycerol stock of E. coli and incubated overnight at 37 oc at 200 rpm.

Fmoc protected amino acids and other chemicals used in peptide synthesis were obtained from Novabiochem (Switzerland). V8 protease and TF A were procured from Pierce Chemical Company (USA), while }-propanol, PMB," Hemin, Dithiothreitol, EDT A were obtained from Sigma Chemical Company (USA). DE52 and CM52 ion exchange resins were purchased from Whatman (UK). Sodium diothinite was procured from Fluka (Switzerland) and Catalase from Boehringer Mannheim (Germany). Carboxypeptidase was obtained from Worthington Biochemical Corporation (USA).

250 mM KCl 55 mM MnCl2.4H20 50 x TAE (1 litre): 242 g of tris base 57.1 ml of glacial acetic acid 100 ml of 0.5 M EDTA Alkaline Lysis Solution 1: 50mM tris-HCl (pH 8.0) 10.0 mM EDTA 50 mM glucose Alkaline Lysis Solution 2: 0.2M NaOH 1% SDS Alkaline Lysis Solution 3: 3.0M Potassium acetate

2.7 mM KCl 10 mM Na2HPO42 mM KH2PO4Tris Buffer Saline (TBS) (10X): 12.1gm Trizma Base 40.0gm NaCl Adjust PH to 7.6; make up the volume to 1 lit with milli Q water. HEPES Buffer Saline: 20 mM HEPES (pH 7.5) 150 mM NaCl Blocking Buffer: 5% fat free milk or 2% BSA in PBST or TBST. Stripping Buffer: 100 mM β-mercaptoethanol 2% (w/v) SDS 62.5 mM Tris-HCl (pH6.7) Luria Broth: 10g tryptone 10g NaCl 5g yeast extract, make up the volume to 1 lit with water. TB buffer for preparation of competent cells: 10 mM PIPES (free acid) 15 mM CaCl2.2H20

2.5 ml of 1.5 M Tris-Cl (pH 8.8) 4.0 ml of 30% acrylamide; bisacrylamide (29:1) mix 50.0 μl of 20% SDS 3.35 ml of milli-Q water 100 μl of 10% APS 10.0 μl of TEMED. 2X SDS loading Buffer: 130 mM Tris-Cl (pH 8.0) 20% (v/v) glycerol 4.6% (w/v) SDS 0.02% bromophenol blue 2% DTT SDS PAGE Running Buffer: 25mM Tris base, 0.2M glycine 1% SDS Western Blot: 1 x Blotting Buffer (2Litres): 25mM tris base, 0.2M glycine 20% methanol Phosphate Buffer saline (PBS): 137 mM NaCl

Whole cell lysis buffer: 20mM Tris (PH 7.5) 150mM NaCl 1mM EDTA 1mM EGTA 1 % triton X 100 2.5mM sodium pyrophosphate 1mM β-glycerophosphate 1mM Na3VO41μg/ml aprotinin, 1μg/ml leupeptin and 1μ.ml pepstatin SDS-PAGE: Stacking Gel Mix (4ml, 5%): 380μl of 1M Tris-Cl (pH 6.8) 500μl of 30% acrylamide ; bisacrylamide (29:1) Mix 15 μl of 20% SDS 2.1 ml of milli-Q water 30 μl of 10% APS 5 μl of TEMED. 12% Resolving Gel Mix (10ml):

Human lung epithelial type II cells (HPLD) were a kind gift from Dr. T Takahashi, Japan. All the cell lines were maintained in 5% CO2 with the recommended media containing 10% FBS (3% FBS for HPLD) and following the standard guidelines.

A549 (human lung adenocarcinoma epithelial) and murine fibroblast cell line NIH3T3 was purchased from American Type Culture Collection. Murine lung epithelial cell line E-9 and E-10 were a kind gift from Dr. L.M.Anderson, (NCI-FCRDC, Frederick, Maryland)

Monoclonal antibody against KRAS were purchased from Merck Research Laboratories, phospho p44/42 (ERK1/2)and total p44/42 (ERK1/2)antibodies were purchased from Cell Signaling Technologies. Anti tubulin antibody was obtained Sgima-Aldrich Chemicals. HRP conjugated anti-mouse and anti-rabbit secondary antibodies were purchased from Bangalore Genei Pvt. Ltd.

All the chemicals used for routine molecular biology work were procured from Sigma-Aldrich Chemicals (St Louis, MO, USA) unless otherwise mentioned. Taq polymerase for PCR and standard DNA markers and protein markers were purchased from MBI Fermentas. Tissue Culture materials like DMEM medium (for A549), Ham’s F-12 medium (for HPLD), Opti-MEM medium, 0.5% trypsin-EDTA, 100X antibiotic-antimycotic, freezing medium, fungizone, 200mM L-glutamine, fetal bovine serum (FBS), Lipofectamin-2000 and TRIzol were obtained from GIBCO BRL (Gaithersburg, Maryland, USA). CMRL medium was purchased from ICN laboratories. M-MLV reverse transcriptase, RNase inhibitor, dNTPs and MgCl2 were obtained from Invitrogen Corporation (Carlsbad, CA). ECL western detection kit and HybondTM- P were purchased from Amersham biosciences (GE Healthcare, UK).

Most chemicals were obtained from commercial sources. The sources for some of the fine chemicals used in this study are given below. Most of the chemicals such as amino acids, antibiotics, sugars, IPTG, ONPG and X-gal were obtained from Sigma Chemical Co. The media components for the growth of bacteria were routinely from Himedia. The materials used in the recombinant DNA experiments such as restriction endonucleases, T4 DNA ligase, DNA polymerases for PCR amplification and DNA size markers were obtained from companies including New England Biolabs and Fermentas. Quiagen or HiPura Kits used for plasmid isolation, purification of DNA fragments. The oligonucleotide primers used in this study were mainly synthesized on order by Ocimum Biosolutions or MWG Biotech Pvt. Ltd

PonceauS stain Instant Blue (Biorad)

Protein loading dye (6X) Tris-Cl (pH 6.8) 300 mM SDS 12% (w/v) Bromophenol blue 0.6% (w/v) Glycerol 60% (v/v) 600 mM β-mercaptoethanol

Stains and Dyes

Antibiotics were used at the following final concentrations (μg/ml): Rich media Minimal media Ampicillin (for plasmids) 100 50 Ampicillin (chromosome) 30 30 Chloramphenicol (for plasmids) 50 25 Chloramphenicol (chromosome) 25 25 Kanamycin 50 25 Nalidixic acid 50 - Rifampicin 100 - Streptomycin 50 100 Streptomycin 100 200 Spectinomycin 50 100 Tetracycline 15 8 Trimethoprim (for plasmids) 60 30 Chloramphenicol 0.1mg/ml The 10 mg/ml chloramphenicol stock in ethanol was used to make 0.1mg/ml solution in water

NP-40 1% Tris 50 mM Sodiun deoxycholate 0.5% SDS 0.1% pH adjusted to 8.0 Running buffer for Western blotting Glycine 14.4g/l Tris base 3.05g/l SDS 1.0g/l Transfer buffer for western blotting Glycine 14.4g/l Tris base 3.03g/l The above salts were dissolved in 800ml of miliQ water and 200ml of methanol was then added. The buffer was chilled before use. PBST for Western blot 10X PBS (1000 ml) Sodium chloride 80 g Potassium chloride 2 g Disodium hydrogen phosphate 14.1 g (Na2HPO4) Potassium dihydrogen phosphate 2.49 g (KH2PO4) 1 l of 1X PBS + 1 ml of Tween-20

TBF-I buffer (200ml) Potassium acetate 0.588 g Calcium chloride 0.249 g Manganese chloride 1.98 g Rubidium chloride 2.418 g 15% Glycerol 30 ml pH adjusted to 5.8 with 1M acetic acid TBF-II buffer (100 ml) MOPS 0.209 g Calcium chloride 1.102 g Rubidium chloride 0.120 g 15% Glycerol 15 ml pH adjusted to 6.5 with 1M potassium hydroxide Acrylamide solution (30%) Acrylamide 29 g Bis-acrylamide 1 g H2O 100 ml Non denaturing polyacrylamide gel (12%) 30% acrylamide 38.6 ml H2O 40.6 ml TBE 20 ml 10% APS 0.7 ml RIPA buffer (Radio Immuno Precipitation Assay buffer): RIPA buffer for bacterial cell lysis Sodium chloride 150 mM

Water to 1000 ml MacConkey lactose agar: MacConkey Agar Base (Difco) 51.5 g Lactose 1% Water to 1000 ml Maloy agar: Tryptone 5 g Yeast extract 5 g NaCl 10 g NaH2PO4 10 g Chlorotetracycline (12.5 mg/ml) 4 ml Water 1000 ml Bacto-agar 15 g After autoclaving, the following solutions were added, ZnCl2 (20 mM) 5 ml Quinaldic acid (10 mg/ml) 10 ml Citrate buffer: (0.1 M; pH 5.5) Citric acid (0.1 M) 4.7 volumes Sodium citrate (0.1 M) 15.4 volumes TBE and TAE buffers: TBE: 90 mM Tris-borate, 2 mM EDTA (pH 8.0) and TAE: 40 mM Tris-acetate, 2 mM EDTA (pH 8.0) were used as standard electrophoresis buffers. TBE and TAE were prepared as 10X and 50X concentrated stock solutions, respectively, and used at 1X concentration

cto-agar 15 g LBON agar: LBON medium1000 mlBacto-agar 15 gLB soft agar: LB medium 100 ml Bacto-agar 0.6 gK-Medium: KH2PO4 1.0 mM FeSO4 0.5 mg/l (NH4)2SO4 1.5 mM MgCl2 0.08 mM Casamino acids 5 g/l Thiamine 2 mg/l pH was adjusted to 7.0 with Tris free base. K-medium is low osmolarity (70 mOsm) medium (Kennedy, 1982). Z broth: LB medium 100 ml CaCl2 (0.5 M) 0.5 ml MacConkey agar: MacConkey Agar (Difco) 51.5 g Water to 1000 ml MacConkey galactose agar: MacConkey Agar Base (Difco) 51.5 g Galactose 1%

Glucose/Glycerol-minimal A 19 amino acid medium: This medium is essentially the same as glucose/glycerol-minimal A medium described above except that all the 19 amino acids (except tryptophan) were added after autoclaving in a final concentration of 40 μg/ml from autoclaved 4mg/ml amino acid stock solutions. Minimal agar: Contained 1.5% Bacto-agar (Difco) in minimal A Medium. The plates were poured after mixing double strength minimal A medium with 4% agar (in water) that had been autoclaved separately. Wherever required, to test polaity relif at lacZ(am) or trpE(fs), meliobose (0.2%) was replaced for glucose and anthranilate at 100 μg/ml (4 mg/ml stock prepared in DMF) was replaced for tryptophan respectively. LB medium: Tryptone 10 g Yeast extract 5 g NaCl 10 g Water to 1000 ml pH adjusted to 7.0 - 7.2 with 1 N NaOH. LBON medium: Tryptone 10 g Yeast extract 5 g Water to 1000 ml pH adjusted to 7.0 - 7.2 with 1 N NaOH LB agar: LB medium 1000 ml

All the media and buffers were sterilized by autoclaving for 15 minutes at 121°C. Media and buffers used in this study are described below. Glucose/Glycerol-minimal A medium: K2HPO4 10.5 g KH2PO4 4.5 g (NH4)2SO4 1 g Sodium citrate, 2H2O 0.5 g Water to 1000 ml After autoclaving the following solutions were added. MgSO4 (1 M) 1 ml Glucose (20%) 10 ml Or Glycerol (80%) 5ml Vitamin B1 (1%) 0.1 ml Amino acids and bases, when required, were added to a final concentration of 40 μg/ml. When growth on other carbon sources was to be tested, glucose was substituted by the appropriate sugar at 0.2%; when used as carbon source, the final concentration of Casamino acids was 0.5%

aStrain DH5α, MC4100 and MG1655 was from our laboratory stock collection. Strains described earlier include GJ3107, GJ3110, GJ3161, GJ3168, GJ3171 (Harinarayanan and Gowrishankar, 2003), and RS353 and RS445 (Chalissery et al., 2007). Strain GJ5147 is an Ilv+ derivative of GJ3073 (Chalissery et al., 2007). Strains GJ6504, GJ6509, GJ6511, GJ6516, GJ6520 and GJ6524 were constructed by S. Aisha (unpublished). Strains GJ5108, GJ5146, GJ5153 were constructed by K. Anupama (unpublished). b Genotype designations are as described in Berlyn (1998). cK7906 strain is described in Zheng and Friedman (1994). d MDS42 strain is as described in Posfai et al. (2006)

Table 2.1 : List of E. coli K-12 strains

genome cloned in a ColEI-based replicon, and obtained from Dr. Manjula Reddy. pHYD2556 is spectinomycin resistant and carries the minimal nusA+ open-reading frame with its native ribosome-binding site between genomic nucleotide co-ordinates 3314061and 3315548 cloned downstream of the ara regulatory region in a pSC101-based replicon, and obtained from Dr. Ranjan Sen. pHYD2557 is chloramphenicol resistant and carries a 2.3-kb PCR-amplified region between genomic nucleotide co-ordinates 3314061 and 3316393 (containing yhbC nusA region with its own promoter) cloned in a pSC101-based Ts replicon, and obtained from Dr. Ranjan.Plasmid DNA preparations were routinely prepared from recA strains such as DH5αand were stored in 10mM Tris-Cl (pH 8.0) plus 1mM EDTA at ─20 ̊C

pWSK30 an Ampicillin resistant vector with pSC101 origin of replication and blue-white screening facility (Wang and Kushner, 1991). pHYD272 is a derivative of pMU575, an IncW-based single copy vector with Trimethoprim resistance marker carrying lacZYA reporter genes under proU promoter (Dattananda et al., 1991). pHYD751 a ColE1 replicon plasmid with ampicillin resistance marker and 2.1kb EcoRI-SalI fragment carrying nusG+cloned into EcoRI-SalI sites of pAM34 vector. The plasmid exhibits IPTG dependent replication (Harinarayanan and Gowrishankar, 2003). pHYD763 is a Ts (maintained at 30 ̊C but not at 37 ̊ or 39 ̊C), CmR, pSC101 derivative carrying 3.8 kb BamHI-SacI fragment of nusG+ cloned into BamHI-SacI sites of pMAK705 (Harinarayanan and Gowrishankar, 2003). pHYD1201 a ColE1 replicon plasmid with ampicillin resistance marker and 3.3kb HindIII-SalI fragment carrying rho+cloned into HindIII-SalI sites of pAM34 vector. The plasmid exhibits IPTG dependent replication (Harinarayanan and Gowrishankar, 2003). pHYD1622 is the derivative of pHYD1201 where the Ampicillin resistance marker has been replaced with Chloramphenicol using Wanner method of gene replacement. Cm gene was amplified from pKD3 plasmid (K. Anupama, unpublished). pHYD1623 is the derivative of pHYD751 where the Ampicillin resistance marker has been replaced with Chloramphenicol using Wanner method of gene replacement. Cm gene was amplified from pKD3 plasmid (K. Anupama, unpublished). pHYD2368 is a derivative of pBAD18 (AmpR) with 1.7 kb fragment encompassing RBS and coding region of uvsW from phage T4gt7 into SacI site of pBAD18 (K. Leela, unpublished). pHYD2554 is a derivative of pMBL18 with ampicillin resistance, carrying the 10-kb EcoRI-HindIII fragment between kilobase co-ordinates 3310.06 and 3320.08 of the E. coli

to CCT mutation leading to a Glutamic acid to Glycine change at the 53rd amino acid and a Threonine to Proline change at the 55th amino acid in the H-NS protein (Willams et al., 1996). pLG-H-NS-I119T is a derivative of pLG-H-NS plasmid with ATC to ACC mutation leading to a Isoleucine to Threonine change at the 119th amino acid in the H-NS protein (Willams et al., 1996). pLG-H-NS-P116S is a derivative of pLG-H-NS plasmid with CCA to TCA mutation leading to a Proline to Serine change at the 116th amino acid in the H-NS protein (Willams et al., 1996). pLG-H-NS-Y97C is a derivative of pLG-H-NS plasmid with TAT to TGT mutation leading to a Tyrosine to Cysteine change at the 97th amino acid in the H-NS protein (Willams et al., 1996). pPMrhoCam is a Ts (maintained at 30 ̊C but not at 37 ̊ or 39 ̊C), CmR, pSC101 derivative carrying PuvII-HindIII fragment containing trxArho+ cloned into PuvII-HindIII sites of pPM103 (Martinez et al., 1996). pTrc99A an expression vector with ColE1 origin of replication and ampicillin resistance marker. Provides IPTG dependent induction of the insert (Amann et al., 1988). pUC19 is a high-copy-number ColE1 based E.coli cloning vector (500-700 copies/cell) with an Ampr selectable marker. It is one of a series of related plasmids constructed by Messing and co-workers and contains portions of pBR322 and M13mp19 (Yanisch-Perron et al., 1985). It carries a multiple-cloning site (MCS) region in the lacZα fragment, and therefore allows for blue-white screening of recombinant clones

pAM34 is a pBR322-derived cloning vector with Ampr and Specr selectable markers. The replication of this plasmid is dependent on the presence of IPTG, the gratuitous inducer of the lac operon (Gil and Bouche, 1991). pBAD18 is an expression vector with a pBR322 derived origin of replication and allows for tightly regulated expression of the genes cloned under the PBAD promoter of the araBADoperon (Guzman et al., 1995). The vector also carries the araC gene, encoding the positive and negative regulator of this promoter. pBluescript II KS (pBKS) is also a high-copy-number ColE1 based cloning vector with Ampr selectable marker and blue-white screening facility (obtained from Stratagene). pCL1920 is a low-copy-number vector with pSC101 replicon (~ 5 copies/cell), that carries streptomycin (Str)/spectinomycin (Spec)-resistance marker (encoded by aadA) and also carries a MCS region within the lacZα that allows blue-white screening to detect recombinants (Lerner and Inouye, 1990). pCP20 pSC101-based Ts replicon, CmR AmpR, for in vivo expression of Flp recombinase (Datsenko and Wanner, 2000). pLG339 is a low-copy-number cloning vector with pSC101 replicon that has a Kanrselectable marker (Stoker et al., 1982). pLG-H-NS is a pLG339 derivative where the hns ORF had been cloned into the EcoRI-SalIsites of pLG339 vector (KanR, pSC101) (Willams et al., 1996). pLG-H-NSΔ64 is a derivative of pLG-H-NS plasmid with AT base pair deletion after codon 63 in the hns gene resulting in a frameshift (Willams et al., 1996). pLG-H-NS-L26P is a derivative of pLG-H-NS plasmid with CTG to CCG mutation leading to a Leucine to Proline change at the 26th amino acid in the H-NS protein (Willams et al., 1996). pLG-H-NS-E53G/T55P is a derivative of pLG-H-NS plasmid with GAG to GGG and ACT

pACYC184 is a medium-copy-number cloning vector (~ 20 copies/cell) with Cmr and Tetrselectable markers. It carries the origin of replication from plasmid p15A (Chang and Cohen, 1978), which is related to and yet is compatible with that of ColE1. This property enables pACYC184 to co-exist in cells with ColE1 plasmid vectors, including all the ones mentioned above

The bacteriophage P1kc was from our laboratory collection and is referred to as P1 throughout this thesis. Phage λcI857 was also from our laboratory collection. Other bacteriophages that were used in this study included the following: (i) λNK1098 carries a Tn10 transposon with a tertracycline (Tet) ressistance marker. (ii) λNK1324 carries a mini-Tn10 transposon Tn10dCm with a chloramphenicol (Cm)-resistance marker, Cmr. The lambda phage vectors above (Kleckner et al., 1991) were used to make random transposon insertions in the chromosome either for the purpose of insertional mutagenesis or for tagging antibiotic resistance markers to point mutations

All the bacterial strains that were used in this study are derivatives of Escherichia coliand their genotypes have been listed in Table 2.1 Bacterial strains were routinely stored on solid agar plates at 4°C and also as thick suspensions in 40% glycerol either at −20°C or at −70°C. Plasmid harboring strains, were reconstructed whenever necessary by fresh transformations

purification of DNA fragments werefrom Qiagen or HiMedia. The oligonucleotide primers used in this study were mainly synthesised by Ocimum Biosolutions or MWG Biotech. The radioactive chemicals were procured from BRIT Mumbai

Chemicals were obtained from commercial sources. Most of the chemicals such as amino acids, antibiotics, sugars, IPTG, ONPG and X-gal were obtained from Sigma Chemical Co. The media components for the growth of bacteria were mostly from HiMedia laboratories. The materials used in the recombinant DNA experiments such as restriction endonucleases, T4-DNA ligase, DNA-polymerases and DNA size markers were obtained from companies including New England Biolabs, MBI Fermentas and Stratagene.RNA isolation chemicals like Reverse transcriptase, trizol, RNA loading buffers and dyes and RNA size markers were obtained from Invitrogen and Sigma. Protein markers were obtained from MBI Fermentas. Kits for plasmid isolation,

Antibiotics were used at the following final concentrations in various media as given inTable 2.4.Table 2.4Concentrations of antibiotics (μg/ml)

Waterto 3 mlTEMED 10 μlDenaturing (urea) sequencing gel (6%) composition10 X TBE 50 ml40% acrylamide 75 mlUrea 210 gm (7 M)Waterto 500 mlThis was filtered through a 0.45/0.22 μ milipore filter.For casting the gel 35 ml of the sequencing gel mixure was mixed with 150 μl10% APS and 25 μlTEMED

Formaldehyde agarose gel(For 50 ml)DEPC treated water 43 mlMOPS buffer 5.3 mlAgarose0.63 gmFormaldehyde2.6 mlThe above mix was boiled without formaldehyde to dissolve agarose and then at around 50ºC formaldehyde was added just before casting the gel.40% Acrylamide solutionAcrylamide39 gmBis-acrylamide 1 gmWater to 100 mlNon denaturing gel composition (50 ml)40% acrylamide solution 5 ml10 X TBE 5 mlH2O 40 ml10% APS 250 μlTEMED 30 μlSDS PAGE gel (12%)For resolving Gel (15 ml):30% Acrylamide solution 6 ml1.5 M Tris-Cl (pH 8.8)3.8 ml10% SDS150 μl10% APS 150μlWaterto 15 mlTEMED 10 μlFor stacking gel (3 ml):30% Acrylamide solution 500 μl1 M Tris Cl (pH 6.8) 380 μl10% SDS 30 μl10% APS 30 μl

Storage buffer for proteinTris-Cl (pH 8.0) 20 mMNaCl 300 mMDTT10 mMGlycerol 40 % Hybridization bufferTris-Cl (pH 8.0) 9 mMEDTA 0.35 mMSample buffer (for SDS-PAGE)Tris-Cl (pH 6.8) 150 mMSDS (20%) 6% v/vGlycerol 30% v/vβ-mercaptoethanol (5%) 15%Bromophenol blue 0.6% (w/v)EMSA binding bufferTris-Cl (pH 7.5) 10 mMNaCl 50 mMEDTA1 mMGlycerol 5 %DTT 5 mMDenaturing gel loading buffer with dyeFormamide 95%EDTA 20 mMXylene Cyanol 0.05 gmBromophenol blue0.05 gmNon denaturing gel loading buffer with dyeTris-Cl (pH 7.5) 250 mMBromophenol blue 0.02%Glycerol 20%

MOPS bufferMOPS 4.16 gm0.5 M EDTA 1.0 mlSodium acetate 0.68 gmWater (nuclease free) to 500 mlIt was filter sterilized and stored in an amber colored bottle. This was prepared as 10 Xstock solution and used at 1 X concentration.INOUE (PIPES) bufferPIPES (free acid) 10 mMCaCl2.2H2O15 mMKCl 250 mMMnCl2.4H2O 55 mMpH was adjusted to 6.7 with 1 N KOH.PIPES gets into solution when the pH is greater than 6.7. MnCl2was dissolvedseparately and added drop by drop with stirring. The pH was adjusted to 6.7 and filtersterilized and stored at –20ºC.Z buffer (for β-Galactosidase assay)Na2HPO416.1 gmNaH2PO45.5 gmKCl0.75 gmMgSO4.7H2O 0.246 gmβ-mercaptoethanol 2.7 mlWaterto 1000 mlpH was adjusted to 7.0 and stored at 4ºC.SDS running bufferTris-base 30.3 gmGlycine144 gmSDS 10 gmWaterto 1000 mlIt was prepared in 10 X concentration and diluted to 1 X for running

Citrate bufferCitric acid (0.1 M)4.7 volumeSodium citrate (0.1 M) 15.4 volumeTE bufferTris-Cl (pH 8.0) 10 mMEDTA 1 mMTBE bufferTris-Borate 90 mMTris-Borate 90 mMEDTA (pH 8.0) 2 mMThis was prepared as 10 X stock solution and used at 1 X concentration.TAE bufferTris-acetate 40 mMEDTA (pH 8.0) 2 mMThis was prepared at 50 X concentrated stock solution. Both TBE and TAE were usedas standard electrophoresis buffers

LB agarLB medium 1000 mlBacto-agar 15 gmZ broth (for P1 transduction)LB medium100 mlCaCl2(0.5 M) 0.5 mlZ agar (for P1 transduction)Z broth 100 mlBacto-agar0.75 gm

Amino acids when required, were added to a final concentration of 40 μg/ml. Whengrowth on other carbon sources was to be tested, glucose was substituted withappropriate sugar at 0.2%.Glucose-minimal A medium, pH 7.4This medium was same as Glucose-minimal A medium described above except for the difference in K2HPO4and KH2PO4which were as mentioned below:K2HPO414.0 gmKH2PO42.7 gmGlucose-minimal A medium, pH 5.8This medium was same as Glucose-minimal A medium described above except for the difference in K2HPO4andKH2PO4which wereas mentioned below:K2HPO41.5 gmKH2PO412.4 gmGlucose /Glycerol-minimal A 19 (18 or 17) amino acidmediumThis medium is essentially the same as glucose/glycerol-minimal A medium described above except that all 19 or 18 or 17 otherthan either Lys or Lys and Arg or Lys and Arg and His amino acids were added after autoclaving at a final concentration of 40μg/ml from autoclaved 4 mg/ml stock solutions.Minimal A agarIt contains 1.5% bacto-agar (Difco) in minimal A medium. The plates were pouredafter mixing double strength minimal A with 3% agarthat had been autoclaved separately.LB mediumTryptone 10.0 gmYeast Extract5.0 gmNaCl 10.0 gmWaterto1000 mlpH adjusted to 7.0 to 7.2 with 1 N NaOH

All media and buffers were sterilised by autoclaving at 121ºC for 15 mins. Mediaand buffers used in this study are given below:Glucose /Glycerol-minimal A mediumK2HPO410.5 gmKH2PO44.5 gm(NH4)2SO41.0 gmSodium citrate, 2H200.5 gmWater to 1000mlAfter autoclaving the following solutions were addedMgSO4(1M) 1 mlGlucose (20%) 10 mlOr Glycerol (80%)5 mlVitamin B1 (1%) 0.1 ml

The primers used in this study are listed in Table 2.3.Table 2.3 Oligonucleotide primersa

bindingsite lie upstream of the MCS to ensure the high level expression of any genecloned in MCS. A stretch of hexa-histidine (His6)-encoding codons followed by stopcodon is incorporated downstream of MCS to give a C-terminally His6-taggedrecombinant protein (EMD Biosciences).6. pBAD18:It is an expression vector with a pMB9derived origin of replication and allows for tightly regulated expression of genes cloned under the PBADpromoter of the araBADoperon (Guzman et al.,1995). The vector also carries thearaCgene, encoding the positive and negative regulator of this promoter.7. pCP20: pSC101-based Ts replicon, chloramphenicol resistant, ampicillin resistant, for in vivoexpression of Flp recombinase (Datsenko and Wanner, 2000)Plasmid DNA preparations were routinely made from recAstrainDH5αandwerestored in 10 mM Tris-Cl (pH-8.0) with 1 mM EDTA at –20ºC. The plasmid constructsused in this study are given in Table 2.2.Table 2.2Plasmid constructs

The plasmid vectors used in this study were as follows:1.pCU22: It is a derivative of pUC19 used to prepare supercoiled DNA for in vitrotranscription where two strong phage fdtranscription terminators flank MCS. This ensures that the transcripts originated from vector based promoters will not interferewith the transcription from the cloned promoter and that the transcript originated fromthe cloned promoter will be terminated after the MCS (Ueguchi and Mizuno,1993).2.pMU575: It is an IncW-based, single-copy, trimethoprim resistance bearingpromoter probe vector. It carries its MCS upstream of a promoterless galK’-lacZfusion. This fusion has the first 58 codons of galKfused to the 8th codon oflacZ, andthe resultant hybrid polypeptide possesses functional β-Galactosidase activity(afterassembly as a tetramer). Translation of the hybrid gene is controlled by the ribosomebinding site of galK. There are stop codons in all the three reading frames between MCS and initiation codon of galKso that there is no interference caused bytranslational read-through from inserts cloned into MCS region. A strong pheRterminator located upstream of the MCS prevents read through from any vector-basedpromoter into the lacZgene (Andrews et al.,1991).3. pTrc99A:It is an expression vector with ColE1 origin of replication and ampicillin resistance marker. It provides IPTG dependent induction of the cloned gene (Amann et al., 1988)4. pCL1920: It is a pSC101-based, low-copy-number vector with spectinomycin and streptomycin resistance marker carrying the MCS in lacZαregion and henceprovides the advantage of screening the insertions using α-complementation (Lernerand Inouye,1990).5. pET21b: It is a ColE1-based, high-copy-number expression vector bearing ampicillinresistance marker. A strong T7 RNAP-recognised promoter and an efficient ribosome

TheE. coli strains used in this study with their genotypes are shown in Table 2.1. All strains other than BL21 (DE3) employed in protein overexpression experiments are derivatives of E. coli K12. Bacterial strains were routinely stored on solid agar plates at 4ºC and also as thick suspensions in 40% glycerol at –70ºC. Plasmid harboring strains were freshly prepared by transformation of the required plasmid. The bacteriophage P1kc from the laboratory collectionwas used for routine transduction tomove a locus from one strain to anotherand is referred to as P1 throughout this thesis.Table 2.1 E. coli strains used in this study

Oligonucleotides/primers used in this study were designed using either free online-tool Primer3 (http://frodo.wi.mit.edu/) or Gene Runner software (http://www.generunner.net/). Oligonucleotides used in this study were commercially

synthesized from MWG Biotech Pvt. Ltd., Bangalore. All primers used in this study are listed in Table 2.3.Table 2.3: List of primers used in this study

10mM EDTA0.1% SDS 1 M ureaToluidine blue staining solution:0.05% Toluidine blue20% Methanol2% GlycerolSolution was prepared in H2O.Destaining solution for polyphosphate gels:20% Methanol2% GlycerolSolution was prepared in H2O.Spheroplast buffer:50 mM Potassium phosphate (pH 7.5)0.6M Sorbitol0.2 X YPD mediumPS(PIPES-Sorbitol)buffer:10 mM PIPES-KOH (pH 6.8)200mM Sorbitol1 X protease inhibitor cocktail (Roche Cat # 04693159001)**To be added fresh before use

Citric-Phosphate buffer:0.5 M citric acid0.5 M dibasic sodium phosphatepH was adjusted to 5.0 with phosphoric acid and filter-sterilized.MES/TEA buffer:1 mM MES(2-(N-morpholino)ethanesulfonic acid)pH was adjusted to pH 5.0 with TEA(triethanolamine).Plasma membrane suspension buffer:50 mM Tris-HCl(pH 7.5)0.1mM EDTA0.1 mM Dithiothreitol 20% GlycerolPolyphosphate extraction buffer:50 mM HEPES (pH 7.2)

Genomic DNAisolation buffersBuffer A:50 mM Tris-HCl10mM EDTA150 mM NaCl 1% Triton-X 1% SDSBuffer B:50 mM Tris-HCl (pH 7.5)10 mM EDTA1.1 M Sorbitol50 mM β-mercaptoethanol(To be added just before use

15% Acetic acidTris-Borate Saline(TBS):25 mM Tris150 mM NaClpH was adjusted to 7.4withHCl.This was prepared as 10 X stock solution and used at 1 X concentration.Blocking and wash buffers(PBS-T and TBS-T):5% Fat-free milk 0.1% Tween-20 Volume was made to 100 ml either with 1 X PBS(PBS-T)or 1 X TBS(TBS-T)

0.02% Bromophenol blue 2% DTT This was prepared as a 4 X stock solution and used at a 1 X concentration.SDS-PAGE running buffer:0.25 M Tris-HCl (pH 8.0) 1.92 M Glycine 1% SDS This was preparedas a 10 X stock solution and used at a 1 X concentration.Coomassie brilliant blue (CBB) staining solution:50% Methanol10% Acetic acid0.1% Coomassie brilliant blue-R250Western blotTransfer buffer:0.25 M Tris-HCl (pH 8.0) 1.92 M Glycine 1% SDS Thiswas preparedas a 10 X stock solution and used ata 1 X concentration.1X Transfer buffer (1litre):200 ml of methanol 100 ml of 10 X transfer buffer 700ml of waterPonceau 3S staining solution:0.25% Ponceau 3S40% Methanol

SDS-PAGE30% Acrylamidesolution29 g Acrylamide1 g Bis-acrylamideDissolved in 100 ml H2O.10% Sodium Dodecyl Sulfate (SDS):10 g SDS in 100 mlH2OResolving gel mix (12%) (15 ml): 4.89 ml H2O6 ml 30% acrylamide:bisacrylamide (29:1) mix3.8 ml 1.5 M Tris-HCl (pH 8.8) 150 μl 10% SDS 150 μl 10% APS 10 μl TEMEDStacking gel mix (3 ml):1.689 ml H2O500 μl 30% acrylamide:bisacrylamide (29:1) mix380 μl 1 M Tris-HCl (pH 6.8) 30 μl 10% SDS 30 μl 10% APS 10 μl TEMEDSDS loading buffer:130 mM Tris-HCl (pH 8.0) 20% (v/v)Glycerol 4.6% (w/v) SDS

Whole cell lysis buffer(Homogenizing buffer):50 mM Tris-HCl(pH 7.5)2 mM EDTA10 mM sodium fluoride*1 mM sodium orthovanadate*1 X protease inhibitor cocktail (Roche Cat # 04693159001)**To be added fresh before use

Buffer C:100 mM Tris-HCl (pH 7.5)10 mM EDTA10% SDSRNA isolation bufferAE buffer: 3 M Sodium acetate0.5 M EDTA(pH 8.0)Phenol:Chloroform:Isoamyl Alcohol (25:24:1)solution:25 volume of Phenol24 volume of Chloroform1 volume of Isoamyl alcholDNA sampleloading buffer:0.25% Bromophenol blue0.25% Xylene cyanol15% Ficoll

15 mM CaCl2.2H2O 250 mM KCl 55 mM MnCl2.4H2O pH was adjusted to 6.7 with 1 N KOH. MnCl2needsto beaddedseparately,drop by drop with stirring, tothe buffer. PIPES goes into solutionwhenpH is greater than 6.7. The solution, after pH adjustment to 6.7 was filter-sterilized and stored at -20ºC.Reagents for yeast transformation:1 M Lithium acetate (LiOAc)50% Polyethylene glycol10 mg/ml Carrier DNADimethylsulfoxide (DMSO)

INOUE transformation buffer:For bacterial DH5α ultra-competent cells preparation10 mM PIPES (free acid)

10 mM Tris-HCl (pH 8.0)1 mM EDTA Tris-Acetic acid EDTA (TAE) buffer:40 mM Tris base 0.5 M EDTApH was adjusted to 8.5 with glacial acetic acid.This was prepared as a 50 X stock solution and used at a 1 X concentration. Tris-Borate EDTA (TBE) buffer:90 mM Tris-borate 2 mM EDTA (pH 8.0) pH was adjusted to 8.3withHCl.This was prepared as a 10 X stock solution and used at a 1 X concentration.Both TAE and TBE were used asstandard gel electrophoresis buffers.HEPES buffer:This was used to prepare YNB medium of different pH.1M HEPESpH was adjusted to 7.5withNaOH.Bufferwas filter-sterilized and stored in an amber-coloured bottle. Citrate buffer(0.1M, pH 5.5):4.7 volume of 0.1 M Citric acid 15.4 volume of 0.1 M Sodium citrate

Phosphate-Buffered Saline (PBS):137 mM NaCl 2.7 mM KCl10 mM Na2HPO42 mM KH2PO4pH was adjusted to 7.3.This was prepared as a 10 X stock solution andused at a 1 X concentration.Tris-HCl buffer:0.5 M TrizmaBase pH was adjusted to7.6 using concentrated HCl.This was prepared as a 10 X stock solution andused at a 1 X concentration.Tris-EDTA (TE)buffer:

0.67% Yeast Nitrogen Base2% DextroseYeast Carbon Base (YCB):1.17% Yeast CarbonBase1% DextroseCAA:0.67% Yeast Nitrogen Base 2% Dextrose0.6% Casamino acids Plates weremade by adding 2% agar

Yeast Extract-Peptone-Dextrose (YPD):1% Yeast extract2% Peptone 2% DextroseYeast Nitrogen Base (YNB)

Luria Bertani (LB):0.5% Yeast Extract1% Tryptone 1% NaCl LB-ampicillinand LB-kanamycin plates:LB medium50 μg/ml ampicillin30 μg/ml kanamycinSuper Optimal Broth (SOB): 0.5% Yeast extract2% Peptone 10 mM NaCl2.5 mM KCl10 mM MgCl210 mM MgSO4

Bacterial medium

antibodies,anti-mouse IgG andanti-rabbit IgG conjugated with horseradish peroxidase (HRP) were obtained from Cell Signaling Technology, USA

All chemicals were purchasedfrom commercial sources. Mediacomponents for bacterial and yeast growthwere obtained from BD (Becton, Dickinson and Company, USA). Other chemicals were purchased from Sigma-Aldrich Co., USA. Materials used in recombinant DNA experiments were primarily obtained from New England Biolabs, Invitrogen, Bangalore Genei and MBI Fermentas. SuperScript™ III first-strand synthesis system was purchased from Invitrogen.MESA GREEN qPCR MasterMix Plus for SYBR®Assay was purchased from Eurogenetec. Kits used for plasmid isolation, PCR product purificationand DNAgelextractionwerefrom Qiagen.Radioactive chemical, ortho-P32-phosphoric acid,wasprocured from BRIT-Jonaki, CCMB, Hyderabad.Anti-Pma1 polyclonal antibody raised against S. cerevisiaePma1 was purchased from Santa CruzInc.,USA. Anti-phospho-p44/42 MAPK (Thr202/Tyr204) was purchased from Cell Signaling Technology, USA. Anti-CPY polyclonal antibody raised against S. cerevisiaeCPY was procuredfrom Thermo Scientific. Anti-Gapdh antibody raised against human Gapdh was purchased from Abcam. Secondary

All C. glabratastrains and plasmids used in this study are listed in Tables 2.1 and 2.2, respectively.Table 2.1: List of yeast and bacterial strains used in this study

Spheroplast resuspension buffer0.1M KCl15 mM HEPES (pH 7.5)3 mM Ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid(EGTA)10% GlycerolPhosphatidylinositol sonication buffer10 mM HEPES (pH 7.5)1 mM EGTA PI3-kinase reaction buffer40 mM HEPES (pH 7.5)20 mM MgCl280 μM ATP5 μCi γ-P32ATPDeveloping solution for thin layer chromatography(120.2 ml)Chloroform –60 mlMethanol –47 mlAmmonia –4.4 mlWater –8.8 ml

SDS-loading buffer was prepared as a 4X stock solutionin H2Oand used at a 1X concentration.SDS-PAGE running buffer0.25 M Tris-HCl (pH 8.0)1.92 M Glycine1% SDSRunning buffer was preparedas a 10X stock solution and diluted to 1X concentration before use.Buffers for Western blotanalysisTransfer buffer (10X stock solution)0.25 M Tris-HCl (pH 8.0)1.92 M Glycine1% SDSTransfer buffer was prepared as a 10X stock solution and diluted to 1X concentration.1X Transfer buffer (1 litre)200 ml of methanol100 ml of 10X transfer buffer700 ml of waterTris-BufferSaline (TBS)25 mM Tris150 mM NaClpH was adjusted to 7.4 with HCl.TBS buffer was prepared asa10X stock solution and diluted to 1X concentration.Blocking and wash buffers (PBS-T and TBS-T)5% Fat-free milk0.1% Tween-20Volume was made to 100 ml with 1X TBS

1 mM sodium orthovanadate1 X protease inhibitor cocktail SDS-PAGE30% Acrylamide solution29 g Acrylamide1 gBis-acrylamideDissolved in 100 ml H2O.10% Sodium Dodecyl Sulfate (SDS)10 g SDS in 100 ml H2OResolving gel mix (12%) (20 ml)6.6 ml H2O8 ml 30% acrylamide:bisacrylamide (29:1) mix5 ml 1.5 M Tris-HCl (pH 8.8)200 μl 10% SDS200 μl 10% Ammonium persulfate(APS)8 μl N,N,N′,N′-Tetramethylethylenediamine(TEMED)Stacking gel mix (5%, 6 ml)4.1 ml H2O1 ml 30% acrylamide:bisacrylamide (29:1) mix750 μl 1 M Tris-HCl (pH 6.8)60 μl 10% SDS60 μl 10% APS6 μl TEMEDSDS loading buffer130 mM Tris-HCl (pH 8.0)20% (v/v) Glycerol4.6% (w/v) SDS0.02% Bromophenol Blue2% DTT

Whole cell lysis buffer (Homogenizing buffer)50 mM Tris-HCl (pH 7.5)2 mM EDTA10 mM sodium fluoride

150 mM NaCl1% Triton-X1% SDSBuffer B50 mM Tris-HCl (pH 7.5)10 mM EDTA1.1 MSorbitol50 mM β-mercaptoethanol (To be added just before use)Buffer C100 mM Tris-HCl (pH 7.5)10 mM EDTA10% SDSAE buffer3 M Sodium acetate(pH 5.3)0.5 M EDTA (pH 8.0)Phenol:Chloroform:Isoamyl alcohol (25:24:1) solution25 ml Tris-equilibrated Phenol24 ml Chloroform1 ml Isoamyl alcholDNA sample loading buffer0.25% Bromophenol blue0.25% Xylene cyanol15% FicollDNA sample loading buffer was prepared in water

Buffer A50 mM Tris-HCl(pH 8)10 mM EDTA

Stripping solutionfor DNA1% SDS0.1% SSCDesired volume was adjusted with sterile water. Alternatively, 0.4 M NaOH was also used to stripthe bound probes fromnylon membranes.HEPES [4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid] buffer1 M HEPESpH was adjusted to 7.5 with NaOH.HEPES was used as a buffering agent for preparing plates of YNB medium of different pH. Buffer was filter-sterilized and stored in an amber-coloured bottle.INOUE transformation buffer10 mM PIPES15 mM CaCl2.2H2O250 mM KCl55 mM MnCl2.4H2OpH was adjusted to 6.7 with 1 N KOH.Yeast transformation reagents1 M Lithium acetate 50% Polyethylene glycol2 mg/ml carrier DNADimethyl sulfoxide (DMSO)Zymolyase cocktail buffer for yeast colony PCR2.5 mg/ml Zymolyase1.2 M SorbitolZymolyase buffer was prepared in 1X PBS

pH was adjusted to 8.5 with glacial acetic acid.TAE buffer was prepared asa50Xstock solution and used at 0.5X concentration.Alkaline denaturing solution for DNAfor membrane preparation0.5 M NaCl0.25 M NaOHVolume was adjusted with sterile water.Denhardt’s solution (50X)1%Ficoll-4001% Polyvinyl pyrollidone1% Bovine serum albuminVolume was adjusted with water and solution was stored at -20°C.Saline Sodium Citrate (SSC) buffer(20X)3.0 M Sodium chloride0.3 M Sodium citrate Volume was adjusted with water and solution was sterilized by autoclaving.Prehybridization Buffer5X SSC5X Denhardt’s solution50% Filtered formamide1% SDSVolume was adjusted with sterile water.Post hybridization wash buffersWash buffer 12X SSC0.1% SDSWash buffer21X SSC0.1% SDS

Phosphate-Buffered Saline (PBS)137 mM NaCl2.7 mM KCl10 mM Na2HPO42 mM KH2PO4pH was adjusted to 7.3 before autoclaving.PBS was prepared as a 10X stock solution and diluted to 1X concentration before autoclaving.Tris-HCl buffer0.5 M Trizma BasepH was adjusted to 7.6 using concentrated HCl.Tris-Cl buffer was prepared as a 10Xstock solution and used at a 1X concentration.Tris-EDTA (TE) buffer10 mM Tris-HCl (pH 8.0)1 mM EDTATris-Acetic acid EDTA (TAE) buffer40 mM Tris base0.5 M EDTA

Luria Bertani (LB)0.5% Yeast Extract1% Tryptone1% NaClSuper Optimal Broth (SOB)0.5% Yeast Extract2% Peptone

Yeast extract Peptone Dextrose (YPD)1% Yeast extract2% Peptone2% DextroseYeast Nitrogen Base (YNB)0.67% Yeast Nitrogen Base2% DextroseFor alternate carbon source utilization experiments, dextrose was replaced withother carbon sourcesviz.,sodium acetate, ethanol, oleic acid, glycerol and citric acid.Yeast Nitrogen Base (YNB) without ammonium sulphate and amino acids0.17% Yeast Nitrogen Base2% DextroseCasamino Acid (CAA)0.67% Yeast Nitrogen Base2% Dextrose0.6% Casamino acidsFor preparing plates, 2% agar was added tothe medium before autoclaving

Table 2.4: List of the oligonucleotides used to confirm deletion of C. glabrataORFs

Table 2.3: List of the oligonucleotides used in the study

Table2.2: List of the antibodies used in the study

Table 2.1: List of strains and plasmidsused in the study

methanol, acetic acid, potassium dihydrogen orthrophosphate, dipotassium hydrogen phosphate, disodium hydrogen orthrophosphate, acetone and citric acid were purchased from Qualigen chemicals. Fluconazole was procured from Ranbaxy.Lysotracker-Red DND 99 and FM 4-64 were obtained from Molecular Probes. Hybond-N and Hybond-P membranes for nucleic acid and protein transfer, respectively, were purchased from Amersham Biosciences. SYBR-green kit for real-time PCR was procured from Eurogentech. Superscipt SS-III RT kit and Pfu polymerase were obtained from Invitrogen. Different restriction enzymes used for cloning and knock-out generation were purchased from New England Biolabs (NEB). High fidelity DNA Pfx polymerase waspurchased fromFinnzymes. Plasmid DNA purification, PCR purification, gel extraction and reaction clean up kits were procured from Qiagen.Medium components for C. glabrataand bacterial culture viz.,yeast extract, peptone, tryptone, cassamino acid hydrolysate, yeast nitrogen base, yeast nitrogen base without ammonium sulphate, yeast nitrogen base without ammonium sulphate and amino acids and yeast carbon basewere purchased from BD (Becton, Dickinson and Company, USA). Animal cell culture media RPMI-1640, DMEM and α-MEM were procured from Hyclone. Fetal bovine serum, glutamine and antibiotics for cell culture medium were obtained from Gibco-Invitrogen

Agarose, phenol, dimethyl sulphoxide (DMSO), sodium acetate, sodium chloride, sodium hydroxide, sodium carbonate, sodium bicarbonate, trizma base, sodium dodecyl sulphate (SDS), formamide, calcium chloride, ethylenediaminetetraacetic acid(EDTA), glycerol, polyethylene glycol, ficoll, diphenyleneiodinium (DPI), methyl methanesulphonate (MMS), camptothecin, hydroxyurea, ammonium persulphate, TEMED, acrylamide, bis-acrylamide, coomassie brilliant blue (CBB), chloroform, formaldehyde, glycine, lithium chloride, lithium acetate, menadione, isopropanol, phorbol myrsityl acetate (PMA), nuclease free water, wortmannin, bafilomycin-A, diethylpyrocarbonate (DEPC), orthrophenylenediamine (OPD), tween-20, acid washed glass beads, trypan blue, Taq DNA Polymease, trisodium citrate dihydrate and uracil were purchased from Sigma Chemicals. β-mercaptoethanol was obtained from GE Biosciences.Protease inhibitor tablets were procured from Roche. Dextrose, sucrose, agar, ammonium sulphate, potassium chloride, caffeine, magnesium chloride and sorbitol were obtained from Himedia.Hydrogen peroxide, hydrochloric acid, sulphuric acid,

Oligonucleotides used in this study were designed either by freely available online tool Primer 3 plus (http://www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi/)or Generunner software. Oligonucleotides were commercially synthesised at MWG Biotech Pvt. Ltd., Bangalore, India. Oligonucleotides used in this study are listed in Table 2.3

All antibodies, their sources, clonality and dilutions used are listed in Table 2.2

All C. glabrataand bacterial strains and plasmids used in this study are listed in Table 2.1

Malachite green reagent

Reaction Buffer

Cell lysis Buffer

DNA staining solution

Fixative

Inoue buffer

DNA loading dye

Agarose gel

TAE

Neutralization solution(Solution III)

Lysissolution(Solution II)

Resuspension solution(Solution I)

Binding Buffer (10X)

EMSA Buffer

Nuclear lysis buffer

Polydeoxy (Inosinate-cytidylate) (Poly dI-dC)

Nuclear extractionbuffer (without protease inhibitors)

Cytoplasmic extractionbuffer (without protease inhibitors)

Blocking buffer: 2% BSA

Permeabilization buffer: 0.2% Triton X100

Fixative : 4% Formaldehyde

Stripping Buffer

Blocking Buffer

TBST

Transfer Buffer

Running Buffer

Stacking and resolving AcrylamidegelsResolving gel (10 ml)

6X protein loading buffer (Laemmlibuffer)

Cell lysis buffer(RIPA Buffer)

Tris Buffered Saline (TBS)

Phosphate Buffered Saline (PBS)

Ammonium persulfate(APS)

Acrylamide (29:1)

Phenylmethylsulfonyl fluoride (PMSF)

Benzamidine

Aprotinin

Leupeptin

NP-40ComponentsFinal concentrationFor 10 mlNP-4010%1mlH2O9ml

Dithiothreitol (DTT)ComponentsFinal concentrationFor 5 mlDTT1.0M0.7725gH2Oq.s

Ethylenediamine tetraacetic acid (EDTA), pH 8.0ComponentsFinal concentrationFor 500 mlEDTA0.5M93.05gH2Oq.sThe pH is adjusted to 8.0 using 10M NaOH

Ethylene Glycol Tetraacetic acid (EGTA), pH 7.0ComponentsFinal concentrationFor 50 mlEGTA0.1M1.902gH2Oq.sThe pH is adjusted to 7.0 using 10M NaOH

Potassium Chloride (KCl)ComponentsFinal concentrationFor 100 mlKCl2M14.91gH2Oq.s

Sodium Chloride (NaCl)ComponentsFinal concentrationFor 100 mlNaCl5M29.22gH2Oq.s

Potassium Chloride (KCl)

HEPES pH 7.9ComponentsFinal concentrationFor 100 mlHEPES1M23.83gH2Oq.sThe pH wasadjusted to 7.9 using 10M NaOH

forpreparation ofregular buffers and solutions viz. Tris, Glycine, SDS, Sodium Chloride, Potassium Chloride, Disodium Phosphate,NP-40, Tween 20, TritonX100, Formaldehyde, Glycerol, Agarose, Acrylamide,Bis-Acrylamide,Ammonium per sulphate (APS), TEMED,BSA, Propidium Iodide, RNase Aetc. were obtained from Sigma(St Louis, MO, USA). PVDF membrane, X –ray films and western blotting detection reagent (ECL prime) were obtained from GE Healthcare (Little Chalfont, UK). Proteaseinhibitor tablets were obtained from Roche (Penzberg,Germany). Anti mouse and anti-rabbit secondary antibodies tagged to HRP (Horse radish peroxidise) were obtained from Bangalore Genei(Peenya, India). Secondary antibodies for Immunofluorescence (anti mouseIgGand anti rabbitIgG) conjugated to Alexa Fluor (488 and 594) from Molecular Probes, Invitrogen and Vectashield mounting medium with DAPI wasobtained from vector laboratories(Burlingame, CA, U.S.A).Antibodies from different sources were used in the present study. The list of different antibodies used in the present thesis is provided in Table 2.1.Table 2.1: List of antibodies used

Media for cell culture (DMEM and Ham’s F12) and foetal bovine serum (FBS) were obtained from Gibco, Invitrogen (Carlsbad, CA, USA). Cell culturereagents such asTrypsin, Phosphate Bufferedsaline (PBS), Antibiotics, Glutamine, etc. were also obtained from Gibco, Invitrogen (Carlsbad, CA, USA). Chemicals for cell culture experiments Aphidicholin, Nocadazole, Polybrene, and Puromycinwere obtained from Sigma (St Louis, MO, USA). Cyclosporine A, MTT (3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide), wortmannin, UO126, SP 600125, cycloheximide, camptothecin, Tacrolimus/FK506 , Tween 20 and Malachite green were obtained from Sigma-Aldrich (St. Louis, MO, USA). Specific calcineurin substrate RII peptide, calmodulin, eIF-2α inhibitor salubrinal, MG-132 and caspase inhibitor z-VAD FMK were obtained from Calbiochem (San Diego, CA, USA). Cytotoxicity detection kit (LDH) was obtained from Roche Diagnostics, (Mannheim, Germany).Live /Dead cytotoxicity assay kit was obtained from Molecular probes, Life technologies, USA.Lipofectamine-2000 and Opti-MEM for transient transfections were also obtained from Invitrogen(Carlsbad, CA, USA).Growth media for bacteria (LB) was obtained from HiMedia laboratories (Mumbai,India). Enzymes used for recombinant DNA experiments (Restriction endonucleases, DNA ligase) were obtained from New England Biolabs (Ipswich, MA, USA). Markers for DNA and protein gels were from Fermentas (Vilnius, Lithuania). Various kits used for macromolecular isolation (Plasmid isolation kit-Mini and midi, Gel extraction kit, PCR purification kit, RNA isolation kit) were procured from Qiagen(Hilden, Germany) or HiMedia (India).Trizol reagent for RNA isolation was obtained from Invitrogen (Carlsbad, CA, USA). BCA protein estimation kit was from Pierce (Rockford Illinois, USA). Cell fractionation kit was obtained from Fermentas (USA). Kitfor TUNEL assay kit wasobtained from Invitrogen(Carlsbad, CA, USA). PCR reagents (PCR buffer, dNTPs, MgCl2, Taq DNA polymerase) were obtained from Fermentas. Polymerasefor long PCRs (AccuTaq) was obtained from Sigma. Reverse transcriptase (SuperScript III) was obtained from Invitrogen. Various chemicals required

Extraction buffer

MTT reagent

6XEMSA sample loading dye

5X EMSA buffer

Native EMSA PAGE

10XBinding buffer

Inoue buffer

6X DNA loading dye

Agarose gel

TAE

Nuclear lysis buffer (without protease inhibitors

Cytoplasmic extraction buffer (without protease inhibitors)

Blocking buffer: 2% BSA

Permeabilisation buffer: 0.2% Triton X100

4% Formaldehyde fixative

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)

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:

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)

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)

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

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

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

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 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

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

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

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

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

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

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

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

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

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

Antibodies used in this study are listed in Table 2.3

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

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

SiRNAWWP2 siRNA described earlier [216]and prevalidated siRNAs for PPM1G (catalog numbers S102658684 and S102658691) were purchased from Qiagen. ShRNAWWP2 shRNA (shRNA1, 5=-CAGGAUGGGAGAUGAAAUAUU-3=;shRNA2, 5= ACAUGGAGAUACUGGGCAAUU-3=)WWP1 shRNA (shRNA1, 5=-ATTGCTTATGAACGCGGCT-3=; shRNA2, ACAACACACCTTCATCTCC-3=)Both WWP2 and WWP1 shRNA were purchased from Open Biosystem.2.1.5Cell linesHeLa cells, HEK293T, and BOSC23celllineswere used in the present study wherever indicated. All the cells werecultured and maintained in RPMI 1640 supplemented with 10% serum and 1% antibiotic (penicillin-streptomycin)at 37° C with 5%CO2.2.2 Buffers and mediaThe buffers and media used in the present study is mentioned in the table 3.Table 3: Buffers and media used in the study

All the primers (sequences)used for cloning the above-mentioned genes are providedin AppendixI

using gateway cloning method (Invitrogen). P73domain deletions were cloned in SFB destination vector. WWP2, WWP1, HACE1, E6AP, and PPM1G were cloned into SFB (S-protein/Flag/streptavidin binding protein (SBP) triple tag), GFP,and Myc mammalian destination vectors using the Gateway cloning technology (Invitrogen). WWP2 domain deletions were cloned into Myc-destination vector. WWP1 domain deletions were cloned into SFB-destination vector. PPM1G domain deletions were cloned into SFB mammalian destination vector using Gateway cloning. Bacterially expressing GST-p73, GST-∆Np73, GST-PPM1G, MBP-WWP1, MBP-WWP2, GST-WWP2, GST-WWP1 and GST-HACE1 were generated by using gateway technology. Ubiquitin WT and all the mutants were cloned into hemagglutinin (HA) mammalian destination vector. Flag-tagged Dvl2was purchased from Addgene. Dvl2 domain deletions were cloned into SFB-destination vectors. All the plasmid constructs generated in the present study are mentioned in table 2.Table 2: Plasmids used in the study

TAp73α and ∆Np73α were kindly gifted by Alex Zaika, Vanderbilt University. Full-length p73 and ∆Np73 were cloned into Myc and HA mammalian destination vectors

All the antibodies used in the present study are mentioned in the table 1.Table 1: Antibodies used in the study

The following chemicalswere used in the present study: Ampicillin, EDTA (USB), dNTPs, Taq DNA polymerase(Fermentas), Pfu DNA polymerase (Stratagene), DpnI (New England Biolabs), Plasmid miniprep, midiprep, and maxiprepkits(Qiagen, and Invitrogen), glycine, EGTA, NaCl, Tris (Fisher Scientific), NH4Cl, acrylamide(SRL), Cisplatin, Doxorubicin, MG132,Cadmium chloride,Nonident P-40, propidium iodide (PI),bis-acrylamide, SDS, TEMED, Ammonium persulphate (APS), CoomassieBrilliant Blue, DAPI, IPTG, kanamycin, Aprotinin, pepstatin, PMSF, -Glycerophosphate, Sodium Fluoride (NaF),Biotin, and DMSO(Sigma), Luciferase assay kit (Promega#1500), Gateway cloning kit, DMEM, FBS, RPMI, Opti-MEM medium,Met-/Cys-DMEM, dialyzed FBS,trypsin-EDTA, L-glutamine, PBS, Lipofectamine 2000, Oligofectamine, (Invitrogen), PEI(Polysciences), milkpowder (Warana), protein G agarose beads, Streptavidin sepharose beads, Glutathionesepharose beads, MBP beads (GE Healthcare), S-protein beads (Novagen/Calbiochem), HA beads(Covance), LB media(Himedia)

Liquid scintillation cocktail5 g PPO (2,5-diphenyloxazol)0.3 g POPOP (1,4-bis (5 phenyl 1,2-oxazole) Benzene Volume was adjusted to 1L with toluene.MUG (4-methylumbelliferyl β-d-glucuronide)extraction buffer1 mM MUG substrate50 mM Sodium dihydrogen phosphate (pH-7.0)10 mM EDTA0.1% Triton X-1000.1% Sodium lauryl sarcosine10 mM β-MercaptoethanolLactophenol solution (100 g)25 g Lactic acid (20.66 ml)25 g Phenol 50 g Glycerol (39.77 ml)These three components were mixed together and 1 volume of lactophenol was added to 2 volumes of ethanol