PI live dead analysis

Cells were grown in 35 mm dishes at 20% initial confluence. At 40-50% confluence, cells were treated with different genotoxic agents as described in Section 2.2.2. Post treatment, cells were washed twice with PBS and replaced with fresh media to allow recovery for different lengths of time. At each time point, cells were harvested and fixed with 70% ethanol at -20°C overnight. Fixation was carried out by adding 70% ethanol drop by drop, while the cells were being vortexed at a low speed.The fixed samples were brought to room temperature, pelleted down at 2000 rpm for 3 min and washed twice with PBS. Cells were stained with PI solution containing 0.1% Triton X-100, 0.2 mg RNase and 20μg propidium iodide and incubated at 37ºC for 30 min in the dark. Samples were analyzed by flow cytometry (FACS ARIA, BD). Data was analyzed usingFACS DIVA (BD) and FlowJo (FLOWJO, LLC) softwares to identify different stages of the cell cycle

Cell cycle analysis by PI staining

Cells were plated at a density of 30,000 cells per well in triplicates in a 24-well plate. After 24 h, cells at a confluency of 30-40%, were treated with different genotoxic agents such as hydroxyurea (0.5 mM), neocarzinostatin (0.25 μg/mL) and mitomycin-C (1 μg/mL) for 12 h. Spent media containing drug was removed and cells were washed gently twice with PBS. Cells were then incubated for different lengths of time ranging from 24 h to 120 h, in fresh complete DMEMto allow them to recover from genotoxic stress. At each time point cell survival was analyzed by MTT assay as described in 2.2.1. Cell survival was expressed as a fold increase in cell population relative to cells treated with drug for 12 h

DNA damage and recovery assay

cells convert MTT to insoluble purple formazan dye crystals which can besolubilized using detergent or DMSO and measured by colorimetry. At each time point mediacontaining MTT dye (250 μg/mL) was added to each well and incubated at 37ºC. After 2 h cells were lysed with 200 μL DMSO for 15 min on a rocker at room temperature to solubilize the formazan and absorbance was monitored at 570 nm using the EnSpire multimode plate reader (PerkinElmer). Doubling time was determined by plotting log [A570] vstime and conducting a linear regression analysis using GraphPad Prism 5

Cells were seeded in 24 well plates at 10% confluence in triplicates and allowed to grow for different lengths of time. Spent medium was replaced with fresh medium every 48 h. Cells were incubated for different lengths of time ranging from 24 h to 120 h, to allow them to grow and cell survival was monitored by the MTT assay. Metabolically active

Cell proliferation assay

3.4 mL H200.63 mL 30% acrylamide solution (acrylamide:bis-acrylamide; 29:1)0.83 mL 1 M Tris (pH 6.8)0.05 mL 10% SDS0.05 mL 10% ammonium persulfate (APS)0.005 mL N,N,N',N'-Tetramethylethylenediamine (TEMED)Resolving gel solution (12%)10 mL3.3 mL H204 mL 30% acrylamide solution (acrylamide:bis-acrylamide; 29:1)2.5 mL 1.5 M Tris (pH 8.8)0.1 mL 10% SDS0.1 mL 10% ammonium persulfate0.004 mL N,N,N',N'-Tetramethylethylenediamine (TEMED)SDS-Running buffer(Tris/Glycine/SDS)25 mM Tris-Cl192 mM glycine0.1% SDSTransfer Buffer25mM Tris-Cl190 mM glycine20% MethanolMTT dyeMTT dye was dissolved in PBS at 5 mg/mL concentration. Filtered through 0.45 μm syringe filters, and stored in dark at 4ºC

Hypotonic lysis buffer10mMTris Cl (pH 7.4)2.5mM MgCl2, 1mM PMSF 0.5% NP-40Hypotonic bufferPrewarmed 0.075 M KCl Fixatives used in this study100% ethanol kept overnight at -20ºCfor immunofluorescence by prelysis protocol70% ethanol kept overnight at -20ºCused for PI based cell cycle analysisMethanol: glacialactetic acid (3:1) for cytogenetic analysis4% Paraformaldehyde-4 g of paraformaldehyde dissolved in 100 mL waterPI staining solutionPBS containing, 0.1% Triton X-1000.2 mg RNase 20μg propidium iodideReagents required for Tris-Glycine SDS-PAGESDS-PAGE 30% Acrylamide solution29 g of acrylamide and 1 g bi-acrylamide (29:1 ratio) dissolved in 100 mL water10% SDS10 g SDS dissolved and 100 mL waterLaemmli buffer40% Glycerol 240 mM Tris/HCl pH 6.8 8% SDS 0.04% bromophenol blue 5% beta-mercaptoethanolStacking gel solution (5%) 5mL

Buffers

Antibodies

Table 2.3: Antibodies used in this study

Click-iT cell proliferation kit (C35002, Invitrogen), Apo-BrdU TUNEL assay kit (A35127, Invitrogen)

Kits

Cell culture reagents: fetal bovine serum (FBS, 26140-079), L-glutamine (25030-081), penicillin-streptomycin (15140-122) and trypsin (25200-056) were from Life Technologies. DNA damaging agents used for this study were obtained from following sources: hydroxyurea (HU; H8627, Sigma-Aldrich), neocarzinostatin (NCS, N9162, Sigma-Aldrich), mitomycin C (M0503, Sigma-Aldrich). Reagents used for cell spreading assays: methyl cellulose (Sigma-Aldrich), fibronectin (F2006, Sigma-Aldrich)andfluorophore conjugated phalloidin (Molecular Probes Inc).Antibiotic selection markers: puromycin (Sigma-Aldrich), G418 (Sigma-Aldrich), transwell inserts (24 well, 8 μm pore size, Costar, Corning), Invasion chambers (BioCoat Matrigel invasion chamber, 24 well, 8 μm pore size, Corning). Other chemicals: Propylene glycol (151957, MP Biomedicals), 4-Nitroquinoline-1-Oxide(4NQO; N8141, Sigma-Aldrich), MTT [(3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide], TNP [N2-(m-(trifluoromethy)lbenzyl)N6-(p-nitrobenzyl)purine],DMSO (dimethyl sulfoxide), ethanol, paraformaldehude, vectashield DAPI (Vector labs),Tris, PMSF, NP-40, PBS, Tween-20, BSA, MgCl2, colcemid, KCl, methanol, glacial acetic acid, giemsa, SDS, sodium bicarbonate (S5761, Sigma-Aldrich),polyfect transfection reagent, crystal violet, propidium iodide, Triton X-100 and PEI were obtained from Sigma-Aldrich. Low melting agarose (Difco), ECL detection system (GE Healthcare)

Chemicals

All animal experiments were conducted as per guidelines provided by the Committee for the Purpose of Control and Supervision of Experiments on Animals, Ministry of Environment, Forest, and Climate Change, Government of India,and these experiments were approved by the Institutional Animal Ethics Committee (Protocol numbers PCD/CDFD/02-version 2 and PCD/CDFD/08). Mice used for this study were housed in the Centre for DNA Fingerprinting and Diagnostics animal facility located within the premises of Vimta Labs, Hyderbad.Ip6k1+/-heterozygous mice were bred to generate age and sex matched Ip6k1+/+and Ip6k1-/-littermates for experiments. Foxn1numice were generated by breeding homozygous males with heterozygous females.These mice were used for in vivotumourigenic assays

Mice

Other plasmids used for lentivirus generation: VSV-G, VSV-GP (gifts from Dr. Renu Wadhwa, AIST, Japan) and psPAX2 (a gift from Dr. Didier Trono, Addgene plasmid # 12260)

Plasmids

Table 2.2:Plasmids expressing shRNAagainst mouse Ip6k2used for generating stable cells in MEFs are listed below

Lentiviral vectors (pLKO.1)encoding various shRNA sequences against human IP6K1and mouse Ip6k2were obtained from Sigma-Aldrichto generate transient and stable knockdowns. shRNA clone IDs and their representation in the thesis are given below.Table 2.1:Plasmids expressing shRNA against human IP6K1used for generating stable cells in HeLa and HCT116

Celllines

The cell lines used in the study are mouse embryonic fibroblasts (MEFs) derived from wild type (WT) and Ip6k1knockout mouse embryos. The MEFs were immortalized with SV40 large T antigen (Bhandariet al., 2008)and single cell derived lines were generated in the lab. Ip6k1knockout MEFs display 70% lower levels of IP7compared with wild type MEFs (Bhandariet al., 2008). Ip6k1-/-MEFs expressing kinase active or inactive forms of IP6K1 were generated in the lab (Rescue MEFs). MEFs were maintained in Dulbecco’s modified Eagle’s medium (DMEM, Life Technologies) supplemented with 10% fetal bovine serum (FBS, Life Technologies), 1 mM L-Glutamine (Life Technologies), 100U/mL penicillin, and 100 μg/mL streptomycin (Life Technologies).Rescue MEFs were cultured in complete DMEM supplemented with G418 (200 μg/mL) as selection marker. HCT116 (colon cancer cells, a gift from Dr. Sagar Sengupta, NII, New Delhi) or HeLa (cervical cancer cells) expressing non-targeting control and shRNA against human IP6K1were cultured in complete DMEM containing puromycin (2μg/mL). The amphotropic Phoenix cells (a gift from Dr. Shweta Tyagi, CDFD, Hyderabad) and HEK293T packaging cells were usedfor generating lentiviral particles containing shRNA against human IP6K1or mouse Ip6k2and were maintained in complete DMEM

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

Jadav Rathan Singh

Investigating the cellular functions of mammalian inositolhexakisphosphate kinase 1 (IP6K1)

Cultures in mid-exponential phasenormalized using A600and solubilizedin 1X sample buffer at 99°C for 5 min were subjected to electrophoresis on 12% sodium dodecyl sulfate (SDS) -polyacrylamide gels. Cell extracts equivalent to 0.04A600(1X) and 0.02A600(0.5X) were loaded and run using Tris-glycine-(SDS) buffer. Separated proteins were electrotransferred to PVDF polyvinyledene difluoride) membrane (Amersham) electrophoretically by a semi-dry method using Bio-Rad apparatus.The transfer was done for 2-3 hrs using a voltage of 75V at 4oC and membrane was probed using anti-FtsZ primary antibody at 1:5000 dilution (rabbit, polyclonal), washed and probed with anti-rabbit IgG conjugated to horseradish peroxidase (HRP) at 1:20000 dilution, as described(Sambrook & Russell, 2001).Membranes were developed with chemiluminescencereagent (Amersham ECL Prime) and visualized with the aid of a chemiluminescence detection system according to the manufacturer’s protocol (Sigma Chemical Co., St. Louis, MO). Quantification of band intensity and subtraction of background was done using Fujifilm Multi Gauge V3.0 imaging system(Image Quant software)

Gel Electrophoresis and Western blotting

Viewing slides under microscope

A drop of immersion oil was put on top of the cover-slip before viewing it under microscope. The cells were viewed at 100X resolution of Nikon Eclipse 80i microscope.Thedifferential interference contrast images of the cells were captured using NIS-Elements D3.0 software also used to find out mean cell size using at least 100 randomly selected cells.Fluorescence images were captured on Zeiss LSM 710 Meta inverted confocal microscope

The slides for microscopy were prepared as described in Dajkovic et al.,(2008)with slight modifications. After wiping the glass slide with ethanol, 200μL of 1% molten agarose was layered on it between two strips of tape and clean cover-slip placed on it to obtain levelled surface. The agarose was allowed to solidify and the cover-slip was carefully removed and5μlof sample was put on top of the agarose and carefully covered with a cover-slip

Preparation of microscopic slides

Fresh overnight cultures grown in LB containing appropriate antibiotics to select for plasmids were sub-cultured 1:100(or lower dilutions for some strains)in the same medium. The cells from these cultures weretaken for microscopy at exponential phase of growth(A600 of 0.5-0.6), as such or after concentrating the cells 10-fold

Sample preparation

Microscopy

The method followed was similar to that describedpreviously with slight modifications (Jinet al., 1992; Schleifet al., 1973).Overnightbacterial cultures were grown in LBand subcultured 1:500 in the same medium in a volume of 20 mlat 30oC.Cultures were induced with 1mM IPTG at A600=0.4. 0.9ml samples were aliquotedat time intervals of 0 sec, 20 sec, 40 sec, 1 min, 1.5 min, 2 min, 2.5 min, 3 min, 3.5 min, 4 min, 4.5 min, 5 min, 5.5 min and 6 min into 0.1ml of 1mg/ml ice cold chloramphenicol and the samples were put on ice. After sampling, 0.5ml of each culture was taken for β-galactosidase assay.Square root of β-galactosidase activity (activity at time Tt−T0) was plotted against time. In the graph, the point of inflection of the curve on the X-axis determines the rate of elongation of RNAP whereas slope represents the promoter clearance, lacZmRNA stability and factors affecting translation of lacZ(Burovaet al., 1995)

RNA polymerase elongation rate measurement

The method followed was as described in Miller (1992). Samples for dot-blotting were prepared by mixing 5μg of RNA (in10μl H20) with 30μl of RNA denaturing solution consisting of 1X MOPS, 7% formaldehyde and 50% deionised formamide. The samples were heat denatured at 65oC for 5 min and mixed with equal volume of 20X SSC. The samples wereloaded into the slots of the dot-blot apparatus (Bio-Rad) containing the membrane (pre-soaked in 20X SSC)and gentle suction was appliedusing Millipore vacuum pump. The slots were rinsed twice with 10X SSC. This was followed by the UV-crosslinking, pre-hybridization, hybridization, washing and exposure of the membrane identical to that done in Northern blotting

Dot-blotting

Non-stringent washes were carried out in 2XSSC and 0.25-0.5% SDS in DEPC water.Stringent washing was done in 1XSSC and 0.5% SDS in DEPC water. Washing was carried out at 55-56oC for 20 minutes. After washing, the blot was covered in the saran-wrap and exposed to the phosphoimager film. After the desired time of exposure, the filmwas then scanned in phosphoimager and the picture saved.The densitometric analysis of the bands was carried out as described in the section 2.2.3.7.Normalization of the signal intensities in northern blotting experiments using probe against tRNA(U73)Arg5was done as follows. The intensity of the tRNA(U73)Arg5signalin the WT or the parent strain in the absence of IPTG was taken as 1 and the relative change in the other strain/growth condition calculated. The value thus obtained was corrected using the change in the corresponding 5S rRNA intensity relative to that in the WT/parent strain in the absence of IPTG

Washingof the membrane, exposure and scanning

For hybridisation,probe was heated at 95oC for 5 minutes and snap-chilled for 5 minutes and then added to the hybridisation bottles containing the blot. Hybridisation was carried out overnight at 50oC.The probes used and their radioactivity counts (in parentheses) were 5s RNA probe (2.5×106cpm), U73 probe (5×106cpm) and lacZ probes (106 cpm)

Hybridisation of the membrane

ii.10% Dextran Sulphateiv.0.5% SDSv.100 μg/mlSalmon Sperm DNAvi.DEPC water5ml of pre-hybridisation buffer was used per blotin 150×35mmhybridisation bottles(Labnet). Salmon sperm DNA was heated at 95oC for 5 minutes and snap-chilled for 5 minutes prior to adding to the rest of the mix. Blot was inserted into the bottle such that it stuck to the wallsand the surface containing the RNA faced the inner side of bottle. Pre-hybridisation was carried out at 50oC for 3 hours in hybridisation chamber(Labnet Problot 12S hybridisation oven)

The pre-hybridization buffer contained the following constituents:i.6X SSCii.5X Denhardt’s solution

Pre-hybridization of the membrane

The RNA was cross-linked onto the membrane after transfer by exposing it to the UV light of 200KJ/cm2 energy in a UV-crosslinker

UV-crosslinking of the RNA

Semi-dry transfer apparatus (Bio-Rad trans blot semi dry transfer cell)was used for the transfer of RNA from the gel to the membrane. The Hybond-N+ membrane from Amersham biosciences was used which was cut as per dimensions of the gel containing the RNA samples. For each transfer 6 pieces of Whatman3mmsheets of the size of the membrane were used. The membrane was soaked for 30-60 minutes in 0.5XTBE before transfer. The transferapparatus was set up as describedby the manufacturer. Transfer was done in 0.5XTBE buffer at 20V, 400mA and 100W for 1.15 hours

Transfer of RNA to the membrane

vii.RNA buffer II from Ambion(1-2X Xylene cyanol + Bromophenol blue)used for loading the samples. RNA isolation for Northern blotting for lacZtranscript was done aftergrowing cultures till A600of 0.6 in LB in the presence or absence of 1mM IPTG at 30oC while for lacZ-lacYʹ-tRNA(U73)Arg5or lacZʹ-tRNA(U73)Arg5transcripts, cultures were grown in LBupto A600of 0.3 and induced with 1mM IPTG for 30 min followed by RNA extraction.30ml of 10% polyacrylamide gels of 1.5mm thickness were cast in the Broviga slab vertical gel electrophoresis apparatus. Gels were polymerizedby the addition of TEMED and APS (1/100th volume of gel mix). The gel was pre-runat 300V for 15-20 minutes prior to loading.Sample preparation for gel loading was done as follows. The normalizedamounts of RNA samplesto be analyzed were mixed with the equal volumes of 2X gel loadingbuffer II(Ambion)making a final concentration of 1X. The samples were then heated at 80 degrees in a thermoblock (eppendorf) for 10 minutes and loaded on the gel when still warm. The gel was run at constant voltage of 300Vfor 3-4 hours till xylene cynol covered 2/3rddistance

The following solutions were used to cast and run denaturing PAGE gels:i.40% acrylamidestock solution ii.7.5M Ureaiii.5X TBEiv.Ammonium persulphate (APS) stock: 10% (w/v) solution made fresh v.TEMED (N,N,N′, N′-tetramethyl ethylene diamine) vi.Gel running buffer (0.5X TBE)

Denaturing polyacrylamide gel electrophoresis of RNA

The method followed was as described in(Lopezet al., 1997)with few modifications. The steps are as described

Northern Blotting

Band intensities in gel autoradiogramswere determined by densitometry with the aid of the Fujifilm Multi Gauge V3.0 imaging system. Equal areas of radioactive bands were boxed and the PSL (Photo stimulated luminescence) values were further considered. Background signal (obtained from equal area as that of the radioactive band but from other part of the gel/blot) is subtracted from the signal intensities obtained from radioactive bands to get the final values

Densitometry

Oligonucleotides and PCR products were end-labelled using phage T4-polynucleotidekinase (PNK, New England Biolabs or Fermentas or Sigma) with 32P-γ-ATP. The radiolabelling reaction mixture (20μl) contained 1X of buffer provided by the company, 10 units of T4-PNK and 40μCi of 32P-γ-ATP. The reaction mix was incubated for 1 hrat 37ºC and the reaction was heat-inactivated at 65oC for 20 minutes. The labelled oligonucleotides and DNA fragments were purifiedby the Qiagen nucleotide removal kit. Labelling efficiency was checkedeither by using Geiger-Muller (GM) counter orusing liquid scintillation counter.For scintillation counting, 1μl of radioactive sample wasadded to the 5ml scintillation cocktail, and radioactivity count was determined in the 32P channel of scintillation counter (Perkin Elmer, Liquid Scintillation analyzer, Tri-Carb 2910 TR, USA). Liquid scintillation cocktail consists of 5g PPO (2,5-diphenyloxazol) and 0.3g POPOP (1,4-bis (5 phenyl 1,2-oxazole) Benzene, adjusted to a volume of 1L in toluene

Radiolabelling of oligonucleotides

DNA sequencing

Automated DNA sequencing on plasmid templates or on PCR products was carried out with dye terminator cycle sequencing kits on an automated sequencer following the manufacturer's instructions byan outsourced sequencing facility

The semi-quantitative reverse transcription-PCR (RT-PCR) involves the synthesis ofcomplementary DNA (cDNA)from RNA. For this, 1μgof RNAwas treated with 1μl (1 unit) DNase I enzyme (Sigma, amplification grade) for20 min to remove DNA contamination. DNase I was inactivated by heating at 70oC for 10 min. Next, 5pmol reverse primer wasadded along with dNTPs and volume made to 10μlwith DEPC-treated water; the mix washeated at 65oC for 5 min and incubated on ice forat least 1 min. The reverse transcription reaction was set up with this mix using the Superscript III RT kit (Invitrogen) as per manufacturer’s protocolto obtain cDNA. The cDNA servedas the template for setting up a PCR for requirednumber of cycles. The samples were finally run on agarose gels

Reverse transcription(RT)-PCR

Concentrations of DNApreparations were estimated by nanodrop or by gel electrophoresis followed by densitometric analysis.Concentration of RNA preparations were estimated by nanodrop

Estimation of DNA and RNA concentrations

Total RNA extraction from E. colicells was doneusing Qiagen RNeasy minikit. Cells were grown to an A600of 0.6 and harvested(amaximumof107cells)at 6000rpm for 5min at room temperature to prevent cells for encountering any stress in cold. Rest of the steps were followed exactly as mentioned in the manufacturer’s protocol. The quality of RNA preparations was assessed following electrophoresison 1.4% agarose-formaldehyde-MOPS gels.Ingeneral,forawild-typestrainRNAyieldwouldbe~0.5-1μg

Isolation of total cellular RNA

For high fidelity PCR, Herculase II fusion DNA polymerase (AgilentTechnologies)was used. Approximately 0.5μg of chromosomal DNAwas used as a template in a 50μl reaction volume

The PCRs were normally performed using Taqpolymerasefrom Roche or Fermentas. Approximately 1-5ng of plasmid or 5-100ng of chromosomal DNA was used as a template in a50μlreaction volume containing 200μM of each dNTP, 20pM each of the forward and reverse primers and 1 unit of Taq DNA polymerase. For colony PCR E. coli cells from a freshly grown plate were resuspended in 10μl of sterile Milli-Q water to get a cell suspension and this was used as a template in a PCR reaction at a final volume of 50μl. The samples were typically subjected to 30 cycles of amplification with the following general conditions: Initial denaturation 95ºC5minutes Denaturation 95ºC 1 minute Annealing 55ºC 1 minute Extension 72ºC 1 minute/kb of DNA template to be amplified Final extension 72ºC 10 minutes

Polymerase Chain Reaction (PCR)

Molecular techniques

Recombineering was performed as described in(Yuet al., 2000)for engineering the linear DNA on the chromosome. The oligonucleotide primers were designed to amplify the DNA cassette to be engineered. Oligonucleotidesused for recombination contained30–50nt homology at the 5ʹ endtothesequences at the target siteand 20nt homology tothe DNA cassette at the 3ʹ end. The DNA cassettefor recombinationwas generated by PCR and would contain30-50 bp homologiesto the target site. A strain with the target DNA and carrying a defective λ-prophage with gam,betaand exo genes (thatfacilitate homologous recombination)under the control of a temperature-sensitive λ cI-repressorwas grown at 30oC. At an A600of 0.4, the culture was shifted to 42oC for 15 minutes to express gam,betaand exo genes. Cells becomecapable ofrecombining linear DNA introduced into the cell by electroporation. 50-100ng ofamplified DNA cassettewas used for electroporation whichwas performed using theBio-Rad Gene Pulser set at 1.8 kV, 25 μF with Pulse controller of 200 ohms

Recombineering

Typically 400-500ng of DNA was used in each ligation reaction. The ratio of vectorto insert was maintained between 1:3 and 1:5 for cohesive end ligation. The reaction was generally performed in 15μl volume containing ligation buffer (provided by the manufacturer) and 0.075 Weiss unit of T4 DNA ligase at 16ºC overnight (14-16 hours)

Ligation of DNA

DNA fragments to be used for specific purposes like ligation or radioactive labellingwere eluted from the agarose gel after electrophoresis. The gel piece containing the desired band was sliced out from the gel and the DNA was purified using commercially available purification kit (Qiagen)for this purpose. The efficiency of elution was determined by checking a small aliquot of DNA sample on the gel

Purification of DNA by gel elution

Around 0.5-1μg DNA was regularly used for each restriction digestion. 2 to 5 units of restriction enzyme were used in the total reaction volume of 20μl containing 2μl of the corresponding buffer supplied at 10X concentration by the manufacturer. The reaction was incubated for 3hours at the temperaturerecommended by the manufacturer. The DNA fragments were visualized after electrophoresis on 0.8 to 1.5% agarose gels. Commercially available DNA size markers were run along with the digestion samples to compare with and to estimate the sizes of the restriction fragments

Restriction enzyme digestion and analysis

The DNA samples were mixed with appropriate volumes of 6X loading dye (0.25%bromophenol blue and 0.25% xylene cyanol and 30% glycerol in water) and subjected to electrophoresis through 0.8 to 1.5 % agarose gel in TAE buffer. The Goodview nucleic acid stain(supplied as 20000X; Beijing SBS Genetech Co. Ltd.) was added to the gel at the time of casting or 6X EZ-Vision One DNA dye(Amresco) was used as loading buffer, both being commercially available non-carcinogenic dyes to aid visualization of bands. The visualization was doneby fluorescence under UV light in a UV transilluminator

Agarose gel electrophoresis

following the manufacturer’s instructions. For genomic DNA, 1ml culture was used for DNA isolationusing Qiagen or Invitrogen kits. The quality of plasmid/genomic DNApreparations was assessed following electrophoresis on 0.8% agarose gels

3ml (for high copy number)or 10 ml (for low-copy number) of cells from an overnight culture were pelleted by centrifuging for 5 minutes at 6000rpm forthe plasmid isolation which was carried out with the commercially available kits (Qiagen or Invitrogen)

Isolation of plasmid and chromosomal DNA

Recombinant DNA techniques

Growth curves were generated to compare the growth rates of E. coli test strains with control strains manually. The appropriate dilutions of the overnight cultures in desired media were made and allowed to grow at required temperature till faint turbidity was visible. At this point samples were collected every 30 minutes until stationary phase was attained. The growth curves weregenerated using Microsoft Excel or SigmaPlot software and growth rates were calculated from the slope of the graph which, in turn, was used to calculate generation time

Estimation of growth rates

β-Galactosidase assay was performed according to(Miller, 1992).Cultures were grown to A600 of 0.4-0.6 from a 1:100 dilution of overnight cultures. Around 0.1-0.5 ml of culture was made up to 1 ml with Z-buffer and lysed with the addition of 100μl of chloroform and 50μl of 0.01% SDS solution. 0.2ml of freshly prepared 4mg/ml ONPG was added to start the reaction and incubated at 28oCtill the colour of the reaction mixture turned yellow. 0.5ml of 1M Na2CO3 was added to stop the reaction and the time duration from initial addition of ONPG to the stopping of the reaction was noted. The absorbance of reaction mix was taken at 420 nm (A420) afterspinning down the mix at 12000rpm for 3 minutes. The A600of the culturesused was also noted. The enzyme’sspecific activity (in Miller units) was calculated using the following equation: β-Galactosidase specific activity (Miller units) = (1000 ×A420) / t × v ×A600Where,‘t’ is the time period in minutes and ‘v’, the volume of culture used in ml

white colonies were recovered and purified to give growth. If the mutation caused synthetic lethality then white colonies (that lack the shelter plasmid) would not be observed since plasmid loss would result in growth arrest. Therefore, lethality was inferred when either white colonies were not recoveredor were recovered but failed to purify further

To determine whether a particular mutation conferred lethality in the ppGpp0or ΔdksAbackground, an assay was devised based on the use of an unstable, easy to cure shelter plasmidpRC7, similar to that described previously(Bernhardt & de Boer, 2004). In the wild-type strain carrying pRC7, this plasmid can be lost at a frequency of 20-30% in the absence of the selection. However, this will not be seen if the plasmid loss leads to cell death. Since the plasmid pRC7 confers a lac+phenotype, in the absence of the selection plasmid loss can be visualized on X-gal IPTG containing plates as white colonies in a Δlac strain whereas the colonies that retain the plasmid will appear blue.In order to carry outsynthetic lethal screen in the ppGpp0or ΔdksAstrains, the spoT or dksAgenes cloned in pRC7 under the control of lacpromoter were used. Theseshelter plasmids,namely,pRCspoT or pRCdksA, respectivelywere transformed into the ppGpp0or ΔdksAstrain. To test the synthetic growth phenotypes, the mutations of the genes to be tested were introduced by phageP1 transductions. The resultingstrains were grown overnight in LBcontaining the antibiotic selection for the shelter plasmid and IPTG for expression of spoTor dksA, subsequently washedin minimal A medium and dilutions(usually 10−5or 10−6) of these cultureswere spreadon X-gal and IPTG containing plates without antibiotic selection for the shelter plasmid. The phenotypes of the white colonies in comparison with the blue colonies were noted. Viability of the strains was inferred when

Blue-white screening for viability or lethality phenotype

or absence of a metabolite or a particular temperature. An EOP of ≤0.01 suggests lethality of the strain on the test medium. For strains carrying IPTG-dependent plasmids, EOP was determined by growing the strains overnight in medium containing IPTG and appropriate antibiotic,and spottingserial dilutions (100or 10–1to 10–6) on +IPTG (permissive) and –IPTG (test) plates to observe growth. Theviability is scored by takingratio of the colony forming units per ml (cfu/ml = No. of colonies × dilution factor × 1000/volume of culture spotted (in μl) obtained on the –IPTG plate to that on the +IPTG plate and determinesthe EOP. Likewise, strains carrying Ts plasmids were cultured overnight at 30°C with the appropriate antibioticand the serial dilutions of this culture were spottedat two temperatures 30°C (permissive) and 42°C (non-permissive or test). The ratio of cfu/mlobtained on the test temperature to that on the permissive temperature determined the efficiency of plating at the test temperature

Efficiency of plating (EOP) is a measure of the ratio of number of colonies (obtained from a given volume of a suitable culture dilution) on a test medium to those on a control or permissive medium, and is a measure of cell viability on the former. It is a very sensitive test and is often used for determining the viability of a strain in the presence

Efficiency of plating (EOP)

C. LBON temperature-sensitivityStrains were streaked on LBON agar plates and after an overnight incubation at42°C, growth was monitored (compared to that on LBON at 30°C as control). Absence of single colony growth was taken to reflect temperature sensitivity. D. In vivotranscription termination phenotypes The rationale for each phenotype is described in the relevant section. SMG-sensitivityThe E. coli relA mutants exhibit SMG-sensitive (SMGs) phenotype,that is,growth-inhibition in the presence of serine, methionine and glycine at 1mM concentration each(Uzan & Danchin, 1978)and is proposed to be a consequence of transcriptional polarity exerted by a frameshift mutation in the ilvG gene on the expression of downstream genes of the ilvGMEDA operon(Lopes & Lawther, 1989).This test was therefore used to distinguish relA+from relA−strains. Growth in the presence of amino acids serine, methionine, and glycine (SMG) was scored on glucose-minimal A plates supplemented with each of the amino acids at 40μg/ml and compared with the growth on non-supplemented glucose-minimal A plates to score for SMG phenotype. galEp3assayThis assay was used to test for relief of transcriptional polarity in the rho and nusG mutants. The galEp3 (galE490*) mutation represents a 1.3kb IS2 insertion in the gal leader region (between the promoter and structural genes of the galETKM operon). The mutation causes transcriptional polarity on the structural genes due to Rho-dependent transcription termination within IS2. In this assay, the gal operon expression in a galEp3 mutant or its derivatives was monitored by usingMacConkey galactose indicator plates (with 1% galactose), where Gal+colonies are red, and Gal−colonies are white. Therefore, the depth of color serves as an indicator of relative levels of gal expression, i.e., the extent of transcriptional polarity relief

Z broth (for P1 transduction) LB medium 100 ml 0.5 M CaCl20.5 ml Buffered LBagarTryptone 10.0 gYeast extract 5.0 gMin A salts1XBacto-agar15.0 gH2O to 1000 ml Buffered Yeast extract agarYeastExtract 5.0 gMin A salts 1X Bacto-agar15.0 gH2O to 1000 ml Yeast extract brothYeast Extract 5.0 g NaCl 10.0 g Bacto-agar15.0 g

A.lacZphenotype lacZ+colonies were distinguished from lacZ–colonies on X-gal containing plate or MacConkey lactose plate. X-gal is non-inducing colourless substrate of β-galactosidase enzyme which upon hydrolysis yields dark blue indolyl group and hence the lacZ+colonies on X-gal plate appear as dark blue colonies. Similarly, on the MacConkey agar plateslacZ+colonies appear dark pink whereas lacZ–colonies remain colourless. B. UV-sensitivityTo check the UV-sensitivity of the strains qualitatively, the strains were streaked on duplicate LB-agar plates and one of the plates was UV-irradiatedwith a 15-W UV-germicidal lamp at a distance of 70cm for 30 seconds. The UV-exposed and unexposed plates were incubated overnight in the dark after wrapping with aluminium foil and then growth was scored. This test could differentiate a recA–strain (UVs) from a recA+strain (UVr)

Scoring for Phenotypes

desired temperaturefor 45 minutes and plated on an appropriate selective medium at various dilutions. An aliquot of cell suspension to which plasmid DNA was not added served as a negative control. B. Inoue method i. Preparation of high efficiency competent cells Competent cells for high efficiency transformation were prepared by the method of (Inoueet al., 1990)with few modifications. An overnight culture of the strain (routinely DH5α) was subculturedinto fresh sterile LB broth in 1:100 dilutions and grown at 18ºC to anA600of 0.55. The cells were harvested by centrifugation at 2500rpm for 10 minutes at4ºC. Thesecells wereresuspended in0.4 volumes of INOUE buffer andincubated in ice for 10 minutes. The cells were recovered by centrifugation at 2500rpm at 4ºC for 10 minutes and finally resuspended in 0.01 volume of the same buffer. Sterile DMSO was added to a final concentration of 7%. After incubating for 10 minutes in ice, the cells were aliquoted in 100μl volumes, snap frozen in liquid nitrogen and stored at –80ºC. ii. Transformation protocolFor transformation, the required number of vials wasthawed on ice and the transformation protocol as described for CaCl2method was employed

A. Calcium chloride(CaCl2)method For routine plasmid transformation, following method which is a modification of that described by(Cohenet al.,1972)was used. An overnight culture of recipient strain was subcultured 1:100 in fresh LB medium and grown till mid-exponential phase. The culture was chilled on ice for 20 minutes, and the steps thereafter performed at 4ºC. 10 ml of culture was centrifuged and pelletwas resuspended in 5 ml of 0.1M CaCl2. After 5 minutes of incubation on ice, the cells were again centrifuged and resuspended in 1ml of 0.1M CaCl2. The suspension was incubated onice for 45 minutes. To the 100μl aliquot of the cellsuspension plasmid DNA (20-200ng in less than 10μl volumes) was added, incubated for 30-40 minutes on ice and given a heat shock for 90 seconds at 42ºC. The cultures were rapidly chilled for 1 minute, mixed with 0.9ml of LB broth and incubated at

Transformation

To 2 ml of fresh overnight culture of recipient strain, 108 pfu equivalent of phage lysate was added and incubated at 37ºC without shaking for 30 minutes to facilitate phage adsorption. The unadsorbed phage particles were removed by centrifugation at 6000 rpm for 5 minutes and the pellet ofbacterial cells was resuspended in 5 ml of LB broth containing 20 mM sodium citrate to prevent further phage adsorption. This was incubated for 25-60 minutes at desired temperaturewithout shaking to allow the phenotypic expression of the antibiotic resistance gene. The mixture was then centrifuged and the pellet was resuspended in 300 μl of 0.1M citrate buffer. 100 μl aliquots were spreadon appropriate antibiotic containing plates supplemented with 2.5 mM sodium citrate. A control tube without addition of P1 lysate was also processed in the same way. In the case of selection of nutritional requirement, the infection mixture was centrifuged, resuspended in 300 μl of 0.1M citrate buffer and plated without phenotypic expression

10–6)were mixed with 0.1 ml of fresh culture grown in Z-broth. After 30 min of adsorption at 37ºC without shaking, each mixture was added on a soft agar overlay of Z-agar plates and incubated overnight at 37ºC. The phage titrewas calculated from the number of plaques obtained on the plates as follows: Phage titre(pfu) per ml = No. of plaques ×dilution factor ×1000/vol.of lysate added (in μl)

Phage P1 lysate preparation by broth method

Phage P1 transduction

EDTA 2 mMThis was prepared at 50X concentration and used at 1X concentration. Both TBE and TAE were used as standard electrophoresis buffers.Gel loading buffer with dye Tris-Cl (pH 7.5) 250 mM Bromophenol blue/ Xylene cyanol 0.02% Glycerol 20%INOUE (PIPES) Buffer PIPES (Free acid) 10 mM CaCl2.2H2O 15 mM KCl 250 mM MnCl2.4H2O 55 mM pHwas adjusted to 6.7 with 1N KOH. PIPES gets into solution when the pH is greater than 6.7. MnCl2 was dissolved separately and added with stirring. The pH was then adjusted to 6.7 and solution wasfilter sterilized and stored at –20ºC.Z Buffer (for β-galactosidase assay) Na2HPO416.1 g NaH2PO45.5 g KCl 0.75 gMgSO4.7H2O 0.246 g H2O to 1000 ml pH was adjusted to 7.0 and stored at 4ºC.Pre-Hybridization Buffer 20X Saline-sodium citrate (SSC)3ml50% dextran sulphate2ml50X Denhardt’s solution1m

20%SDS250 μl10 mg/ml Salmon sperm DNA100 μlDEPC waterto 10mlHybridization Buffer Same as pre-hybridisation buffer but contains the radio-labelled probe.SDS sampleBuffer(1X)Tris-HCl, pH 6.850mMGlycerol10%EDTA12.5 mM SDS2%Bromophenol blue0.02%β-mercaptoethanol1%Running buffer for western blottingGlycine14.4 g/lTris base3.05 g/lSDS1.0 g/lTransfer buffer for western blottingGlycine14.4 g/lTris base3.03 g/lThe above salts were dissolved in 800 ml of milliQ water and 200 ml of methanol was then added. The buffer was chilled before use.TBST buffer for Western blot10X of TBS (1000ml)Sodium chloride80 gPotassium chloride2 gDisodium hydrogen phosphate(Na2HPO4)14.1 gPotassium dihydrogen phosphate(KH2PO4)2.49 gMilliQ waterto 1000m

0.3 ml of overnight culture of the donor strain in Z-broth was mixed with 107plaque forming units (pfu) of a stock P1 phage lysate prepared on strain MG1655. Adsorption was allowed to occur at 37ºC for 30 minutes and the lysate was prepared by broth method. To 0.3 ml of infection mixture, 8-10 ml of Z-broth was added and incubated at 37ºC with slow shakinguntil the visible lysis of the culture occurred (in 4-6 hours). The lysate was treated with 0.2ml of chloroform, centrifuged and the clear lysate wasstored at 4ºC with chloroform.Quantitation of Plaque forming units (pfu)To quantitate the titreof P1 lysate preparation, titration was done using P1 phage sensitive indicator strain such as MG1655. 100 μleach of dilution of phage (typically 10–5,

Z broth (for P1 transduction) LB medium 100 ml 0.5 M CaCl20.5 ml Buffered LBagarTryptone 10.0 gYeast extract 5.0 gMin A salts1XBacto-agar15.0 gH2O to 1000 ml Buffered Yeast extract agarYeastExtract 5.0 gMin A salts 1X Bacto-agar15.0 gH2O to 1000 ml Yeast extract brothYeast Extract 5.0 g NaCl 10.0 g Bacto-agar15.0 g

LB mediumTryptone 10.0 g Yeast Extract 5.0 g NaCl 10.0 g H2O to 1000 ml pH was adjusted to 7.0 –7.2 with 1 N NaOH.LB agar LB medium 1000 ml Bacto-agar 15.0g LB soft agar LB medium 100 ml Bacto-agar 0.6 g

Genetic Techniques

Chemicals were obtained from different 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 Difco laboratories and Himedia. The materials used in the recombinant DNA experiments such as restriction endonucleases, T4-DNA ligase, DNA polymerases, polynucleotide kinaseand DNA size markers were obtained from companies including New England Biolabs, Invitrogen, Promega, Bangalore Genei, Sigmaand MBI Fermentas. Kits used for plasmids and genomic DNA isolation,purification of DNA fragments and PCR amplification were obtained from Qiagenand Invitrogen. High fidelity enzymes for PCR amplification were purchased from Sigma. The oligonucleotide primers used in this study were synthesized by Xcelris genomics orMWG Biotech. The radioactive chemicals were procured from Jonaki

The antibiotics were used at the below concentrations (μg/ml) unless otherwise stated

Chemicals

Antibiotics

EDTA 2 mMThis was prepared at 50X concentration and used at 1X concentration. Both TBE and TAE were used as standard electrophoresis buffers.Gel loading buffer with dye Tris-Cl (pH 7.5) 250 mM Bromophenol blue/ Xylene cyanol 0.02% Glycerol 20%INOUE (PIPES) Buffer PIPES (Free acid) 10 mM CaCl2.2H2O 15 mM KCl 250 mM MnCl2.4H2O 55 mM pHwas adjusted to 6.7 with 1N KOH. PIPES gets into solution when the pH is greater than 6.7. MnCl2 was dissolved separately and added with stirring. The pH was then adjusted to 6.7 and solution wasfilter sterilized and stored at –20ºC.Z Buffer (for β-galactosidase assay) Na2HPO416.1 g NaH2PO45.5 g KCl 0.75 gMgSO4.7H2O 0.246 g H2O to 1000 ml pH was adjusted to 7.0 and stored at 4ºC.Pre-Hybridization Buffer 20X Saline-sodium citrate (SSC)3ml50% dextran sulphate2ml50X Denhardt’s solution1ml

20%SDS250 μl10 mg/ml Salmon sperm DNA100 μlDEPC waterto 10mlHybridization Buffer Same as pre-hybridisation buffer but contains the radio-labelled probe.SDS sampleBuffer(1X)Tris-HCl, pH 6.850mMGlycerol10%EDTA12.5 mM SDS2%Bromophenol blue0.02%β-mercaptoethanol1%Running buffer for western blottingGlycine14.4 g/lTris base3.05 g/lSDS1.0 g/lTransfer buffer for western blottingGlycine14.4 g/lTris base3.03 g/lThe above salts were dissolved in 800 ml of milliQ water and 200 ml of methanol was then added. The buffer was chilled before use.TBST buffer for Western blot10X of TBS (1000ml)Sodium chloride80 gPotassium chloride2 gDisodium hydrogen phosphate(Na2HPO4)14.1 gPotassium dihydrogen phosphate(KH2PO4)2.49 gMilliQ waterto 1000ml

1litreof 1X TBS +1 ml of Tween-2040% Acrylamide solution (29:1) Acrylamide39 g Bis-acrylamide 1 g H2O to 100 ml7.5M Urea 10%acrylamidecomposition 40% Acrylamide 12.5mlUrea22.5g5X TBE 10ml DEPC treated H2O to 50ml The gel mixtures were filtered through a 0.45 μ Millipore filter before adding APS and TEMED

Citrate Buffer Citric Acid (0.1 M) 4.7 volumesSodium citrate (0.1 M)15.4 volumesTE Buffer Tris-Cl (pH 8.0) 10 mM EDTA 1 mMTBE Buffer Tris-Borate 90 mM EDTA 2 mMThis was prepared as 5X solution and used at 0.5X concentration.TAE Buffer Tris-Acetate 40 mM

Buffers and solutions

H2O to 1000 ml LBON medium (LB medium without NaCl) Tryptone 10.0 g Yeast Extract 5.0g H2O to 1000 ml pH was adjusted to 7.0-7.2 with 1N NaOH. LBON agar LBON medium 1000 ml Bacto-agar 15.0gMacConkey Agar MacConkey agar (Difco) 51.5 g H2O to 1000 ml

LB mediumTryptone 10.0 g Yeast Extract 5.0 g NaCl 10.0 g H2O to 1000 ml pH was adjusted to 7.0 –7.2 with 1 N NaOH.LB agar LB medium 1000 ml Bacto-agar 15.0g LB soft agar LB medium 100 ml Bacto-agar 0.6 gZ broth (for P1 transduction) LB medium 100 ml 0.5 M CaCl20.5 ml Buffered LBagarTryptone 10.0 gYeast extract 5.0 gMin A salts1XBacto-agar15.0 gH2O to 1000 ml Buffered Yeast extract agarYeastExtract 5.0 gMin A salts 1X Bacto-agar15.0 gH2O to 1000 ml Yeast extract brothYeast Extract 5.0 g NaCl 10.0 g Bacto-agar15.0 g

All media and buffers were sterilized by autoclaving at 121ºC for 15 minutes. Media and buffers used in this study are given below: Glucose Minimal A medium Minimal A salts(1X)K2HPO410.5g KH2PO44.5g (NH4)2SO41.0g CH3COONa.2H2O 0.5g H20 to 1000 ml After autoclaving the following solutions were addedto Min A salts: MgSO4(1M)1 ml Glucose (20%) 10 ml Vitamin B1 (1%) 0.1 ml Amino acids when required were added to a final concentration of 40 μg/ml or casaminoacids were added at a concentration of 0.2% whenever needed.Minimal A agar It contains 1.5% bacto-agar (Difco) in Minimal A medium. The plates were poured after mixing double strength Minimal A with 3% agar.M9 minimal medium(1X)Na2HPO4•7H2O7.0 gKH2PO43.0 gNaCl0.5gNH4Cl1.0 gH20to 1000 mlSterilize the solution by autoclaving.Glucose-M9 minimal medium was madein asimilarwayto that of Glucose Minimal A medium

Media

Table 2.3: Oligonucleotide primers

Primers

Table 2.2 List of plasmids used in this study

Plasmids

a-Genotype designations are as in (Berlyn, 1998).b-Strains DH5α, MC4100andMG1655 were from the laboratory stock collection.FRT-indicates FLP recognition target, a sequence that is recognized by FLP recombinase and is left as a scar at the indicated sites after excision of an antibiotic marker that is flanked by the FRT sites

The E. coli strains used in this study with their genotypes are shown in the table below. Bacterial strains were routinely stored on solid agar plates at 4ºC and also as thick suspensions in 20% glycerolat –80ºC. Plasmid harbouring strains were freshly prepared by transformation of the required plasmid. The bacteriophage P1kc was used for routine transduction to move a locus from one strain to another and is referred to as P1 throughout this thesis.Bacterial Strains Table 2.1: E. coli strains used and constructed in this study:

Centre for DNA Fingerprinting and DiagnosticsHyderabad, India

Sheo Shankar Pandey

Role of iron and secreted virulence functions inXanthomonasvirulence

Studies on the physiological roles of basal (p)ppGpp and DksA in Escherichia coli

Centre for DNA Fingerprinting and DiagnosticsHyderabad 500001, India

Aanisa Nazir