The reaction mixture contained 0.2 mL of enzyme sample, 0.3 mL of buffer and 0.5 mL of p-nitrophenyl-β-D-glucopyranoside (1.0 mM) prepared in 100 mM buffer as the substrate. The reaction was terminated after 30 min of incubation at 70 °C by adding 2 mL of sodium carbonate-bicarbonate buffer (0.1 M, pH 10.0). The liberation of p-nitrophenol was measured at 400 nm and its yield was determined using a standard curve of p-nitrophenol (1-10 μg mL-1) prepared in sodium carbonate-bicarbonate buffer

β-Glucosidase

Overnight grown cultures of E. coli DH5α, E. coli BL21 (DE3), E. coli XL1blue cells with and without constructs were preserved in 80 % v/v glycerol

MAINTENANCE OF THE RECOMBINANT STRAIN

incubated on ice for 5 min, buffer N3 (350 J.!l) was added to the mixture and the tube was iriverted 4-6 times until mix appeared cloudy. Cell debris was removed by centrifugation at 12000 x g for 10 min and the supernatant was applied to QIAprep spin columns. Columns were centrifuged at 12000 x g for 1 miri and the flow through was discarded and columns were washed using 750 J.!l of 70% ethanol and centrifuged, at 12000 x g for 1 min. Additional centrifugation was performed to remove the residual ethanol. The columns were placed in a 1.5 ml microfuge tube and DNA was eluted with autoclaved water or 1 mM Tris-HCI (PH 8.0).

Plasmid DNA was extracted using commercially available kit (Qiagen, Germany) as per manufacturer's instructions. For a miniprep, bacterial cell pellet from 5ml freshly grown culture were resuspended in 250 III buffer PI containing RNaseA in a microfuge tube, followed by lysis in 250 III of buffer P2. After the tube was

Plasmid DNA Isolation

700 mM NaCl, 12.5 mM CaCh, pH 7.4). 5 J.!L of Annexin-V conjugated to Alexa fluor 488 and 1 J.!L of working solution of PI (100 Jlg/mL) were added to the 100 J.!L cell suspension. Cells were incubated for 15 min at room temperature. Following this, 400 J.!L of IX Annexin binding buffer was added to dilute the sample. The samples were placed on ice. The fluorescence was measured by flow cytometry in FL 1 and FL2 channels for Annexin-V-Alexa fluor 488 and PI fluorescence respectively.

The Vybrant apoptosis assay kit was used to perform Annexin-V/PI staining as described previously (3). The assay is based on the principle that apoptotic cells show loss of membrane asymmetry by exposing phosphatidylserine on the outer surface of the plasma membrane for which Annexin-V, a phosphlipid binding protein, shows high affinity. Hence, Annexin-V conjugated to Alexa fluor 488 binds to phosphatidylserine exposed on apoptotic cells, while propidium iodide binds to nucleic acids of all non-viable cells including necrotic and apoptotic cells. Thus, flow-cytometric analysis of Annexin-V /PI stained cells reveals distinct cellular populations, with the viable cells displaying little or no fluorescence; the early apoptotic cells show green fluorescence of Annexin-V conjugated to Alexa fluor 488; the late apoptotic cells display both green and red fluorescence, while necrotic cells show red fluorescence. The cells after appropriate treatment were harvested by centrifugation at 250 x g for 5 min and were given two washes with ice-cold 1X PBS following which they were resuspended in 100 J!L of ice-cold 1X Annexin binding buffer (50 mM HEPES,

Assay for detection of apoptosis by Annexin-V /PI staining

function of resolution. The displacement for an isotropic B-value is related to the displacement u by the equation B = 8;(u2). The isotropic B-value assumes equal movements in all the directions. However, the vibration of an atom need not be the same in all the directions, and in such a case motion is described by anisotropic displacement parameter. In this formulation the motion is described by an ellipsoid that can be rotated in any direction. The entire anisotropic displacement can be described in terms of six elements: UIJ, U22 and U33 specify the magnitude of movement in three axis and U 12, U t3 and U23 specify the rotation off the principal axis. Anisotropic displacement parameters can be converted to the isotropic equivalent by the formula Biso = 8;(Ull+l'22+U33). The B-values are restrained during refinement. Atoms that are bonded to each other influence each other's motion. B-values are restrained in such a manner that the average difference in the B-values of bonded atoms is kept to a target value. The B-values should vary smoothly along the protein chain and within the side chain. The usual target restraint for adjacent bonded main chain atom is 1.0 and for side chain the target value is 1.5 since one end of the side chain is free, ensuring the higher gradient. Similarly B-values can be graded for the one to three members of a bond angle. For main chain angles the target value is 1.5 and for the side chain angle the value is set to 2.0. Like rigid body refinement, it is done at the early stages of the refinement process. Refinement of the atomic B-factors is a bit tricky and is carried out in later stages of refinement.

The individual atoms were then refined by several cycles of conventional positional refinement, which uses the conjugate gradient minimization method. The proper weight term called Wa was calculated which was used for subsequent positional refinement (Brunger et al., 1987). In case of CNS this value was set to -1 and the program itself calculated these weights. The refinement was started using data in the range 50 A -4.0 A and higher resolution data were added in a stepwise fashion. After complete data had been added, the F0-Fc and 2F0-Fc electron density maps in addition to the composite omit map were calculated and displayed on an HP xw8400workstation (Hewlett-Packard Company, U.S.A.) using Coot (Emsley P, 2004 ). The electron density map was examined in the context of the model and the regions of the map where the electron density was not satisfactory or the model did not fit the density were identified. Using Coot, the residues were mutated to the sequence of the molecule of interest and wherever required, moved locally to correspond to the visible density. This was followed by refinement to check if the changes made could be accepted or not. Depending on the resolution to which X-ray data was available, anisotropic or individual B-factors were also refined. This process of model building and refinement was carried out iteratively until all the differences in sequence with the probe model had been accounted for and there was no ambiguity in the fit between the model and the electron density. Water molecules were included in the model using the 'water pick' program available in CNS only after a sufficient level of refinement had been achieved. This was followed by visual examination of the waters to avoid inclusion of spurious water molecules. The B-value is the measurement of the displacement of an atom from thermal motion, conformational disorder and static lattice disorder. This vibration will smear out the electron density and will also decrease the scattering power of the atom as a

sum runs over all the reflections in your data set, and k is a scale factor needed to put the Fe on the same scale as the Fobs· A model consists typically of five parameters for each atom: x,y,z, B, and Q. The triplet (x,y,z) specifies the position of each atom in an orthogonal coordinate system. B is the B-factor or temperature factor of each atom, and it is related to the thermal motion of the atom. B-factor also contains information about other types of "disorder" including errors that you are being made while constructing and refining model. Q is the occupancy and it is the fraction of time that the atom spends at position (x,y,z). Typically, Q=l. If one has data better than about 1.8 A, then occupancies between zero and one are sometimes used. Refinement procedures for antibodies involve two basic procedures, rigid body refinement followed by positional refinement. Rigid body refinement is used to refine the results obtained from MR in terms of orientation and position of the starting model in the unit cell. Positional refinement is used to refine the positions of individual atoms in space. Both conventional R-factor (Rcryst) and the free R-value (Rfree) (BrUnger, 1992) were used to monitor the progress of refinement. 10% of the reflections were set aside at random to monitor the Rfree during refinement. Rigid body refinement was carried out to further refine the positioning of the probe molecule in the target unit cell. The probe models that gave the highest correlation coefficients were thus subjected to rigid body refinement. Refinement was initially done using data in the range of 50 A -4 A; thereafter data up to the maximum available resolution were added in a step wise manner. The Fab molecule can be defined as an assembly of four domains, the VH, VL, CH and CL. Consequently, rigid body refinement where these domains were considered as discrete rigid units was carried out.

This section describes the detailed refinement strategies used for structure determination. The structures presented in the thesis were refined using CNS (Brunger, 2007; Brunger et al., 1998). The refinement of structures obtained after molecular replacement was done using Crystallography and NMR system (CNS) suite of programs (BrUnger et al., 1998) based on the refinement of crystal structures using Cartesian (BrUnger et al., 1987) or torsion-angle molecular dynamics (Rice and BrUnger, 1994). This task file automatically computes a cross-validated crA estimate, determines a weighting scheme between the X-ray refinement target function and the geometric energy function, refines a flat bulk solvent model (Jiang and Brunger, 1994) and an overall anisotropic B-value of the model by least squares minimization, and subsequently refines the atomic positions. Available target functions include the maximum-likelihood functions MLF, MLI and MLHL (Pannu et al., 1998). Refinement is an iterative process in which the atomic model is modified, structure factor amplitudes are calculated from the modified model, and the agreement between these calculated structure factor amplitudes (IFcl) and the experimental or observed ones (IF ~bsD is determined. The goal is to find the model that produces the best agreement between lfohsl and lfcl. Refinement is a problem of finding the minimum of a function that mathematically expresses the agreement between IFobsl and lfcl. This function is called a target function. A commonly used target function is the crystallographic residual: SUM {(IFobsl -k1Fcl)2}, where the

Structure refinement using the Crystallography and NMR system (CNS) suite

A control reaction was done with the control annealed DNA lacking the oligonucleotide. The constituents were mixed and incubated on ice for 2 min. afterwards, at room temperature, for 5 min. The reaction was further carried out at 37 °C for 2 h followed by heating at 70 °C for 10 min. 12.5 J.tl of the reaction products were analyzed on an agarose gel along with annealed samples to check the complementary strand synthesis. The samples were diluted I 0-times with water and 5 J.ll of the diluted sample was used to transform 50 J.ll of E. coli host strain, DH5a cells. The suspension was pl~!ed on 2 LB agar plates containing ampicillin. Single colonies were picked, grown in liquid culture and miniprep screening of DNA was done to select the positive clones.

Annealed DNA 10.0 J.ll I OX synthesis buffer 2.5 J.ll 1 OX ligation buffer 1.0 J.ll dNTP mix (100 mM) 1.0 J.ll ATP (100 mM) 0.25 J.ll ~ T7 DNA polymerase (5 U/J.tl) 0.5 J.ll T4 DNA ligase (400 U/J.tl) 0.5 J.ll H20 9.25 J.ll Total 25.0 J.l

Titration of uracil containing template: The crude preparation of the phagemid DNA (1 ml aliquot), was used to titrate the uridine incorporation in the template. The strains CJ236 (ung-duf) and DH5aF'(ung+ dut+) of E. coli were transformed with the diluted template DNA The CJ236 cells, plated in the presence of ampicillin and chloramphenicol while DH5aF' cells, plated in the presence of ampicillin alone, were grown overnight on LB agar plates. The good incorporation of uridine gave no colonies or very few colonies in DH5aF' cells whereas with CJ236 several colonies were obtained. A ratio of 103-104 between the number of colonies in CJ236 to that in DH5aF' cells was considered ideal for an efficient incorporation of uridine. Phosphorylation of the mutagenic oligonucleotide: The components of a standard reaction to carry out the phosphorylation are described below Oligonucleotide (180 nmoVml) 1.0 J.ll 1 OX Kinase buffer 2.5 J.ll 10mMATP 1.0 J.ll 10 mM spermidine 0.25 J.ll 100mMDTT 1.25 J.ll T4 polynucleotide Kinase 0.5 J.ll H20 18.5 J.ll Total 25.0 J.LI The constituents were . mixed thoroughly, incubated at 37 °C for 30 min. and subsequently, the enzyme was denatured by heat inactivation at 70 °C for 10 min. Annealing of the mutagenic oligonucleotide: 750 ng (approximately) of the uracil containing single stranded template and 1 J.L) of the phosphorylated· oligonucleotide were taken in IX annealing buffer making up the total reaction volume 20 J.LI. A control reaction, was also carried out simultaneously, lacking the oligonucleotide. The contents were mixed by vortexing and incubated at 95 °C for 10 min. in a water bath. The reaction mixtures were further incubated at 80 °C for I 0 min. in a heat block and the heat block was transferred to ambient temperature, cooled slowly to about 30 °C over a period of 30-60 min. Complementary DNA strand synthesis: The oligonucleotide annealed uracil containing template was used for complementary strand synthesis in the following reaction.

Site-directed mutagenesis was done according to the method employed by Kunkel et al., 1987. Preparation of Uracil containing phagemid: E. coli strain CJ236 was transformed with the required template DNA and grown on LB plates containing the antibiotics, ampicillin 100 Jlg/ml and chloramphenicol 30 Jlg/ml (stock solution made in alcohol). Further, the plates were incubated at 37 °C for 12 h and a single colony was picked from the center of the plate, inoculated in 5 ml LB containing ampicillin and chloramphenicol. The liquid culture was grown at 37 °C overnight with vigorous. shaking. About 500 Jll of the culture was diluted 40-times with LB containing ampici11in and chloramphenicol and grown at 37 °C with vigorous shaking (200 rpm) upto an OD600 of 0.25-0.3. The speed of shaker was reduced to 100 rpm and the culture was left for 30 min. for the F pilus to grow. Afterwards, it was infected with VCS Ml3 helper phage at an MOl of 1:20. The cells were grown for 30 min. in a stationary culture to allow the phages to infect, followed by slow shaking ( 100 rpm) for one hour. Subsequently, the culture was diluted 10-times with 2X YT medium containing ampicillin and chloramphenicol and grown in the presence of 0.25 Jlg/ml uridine and 50 Jlg/ml kanamycin at 37 °C overnight with vigorous shaking. The following day, the culture was chilled on ice for 10 min. and centrifuged at 12,000 rpm for 10 min. at 4 °C in a Sorvall RC5C centrifuge using a GSA rotor. The pellet was discarded and the supernatant was centrifuged again in fresh GSA bottles. A small aliquot of about 1 ml from the supernatant was saved for titration and the . precipitation of the single stranded phagemid was carried out using 0.15 volume of 16.67% PEG in 3.3 M NaCl followed by incubation on ice for 4 h. J'he mixture was centrifuged at 12,000 rpm at 4 °C for 30 min. using a GSA rotor and the pellet was resuspended in 3 ml TE buffer. The suspension was centrifuged at 15,000 rpm at 4 °C for 10 min. using a SS34 rotor. The supernatant was ultracentrifugated at 100,000 g at 4 °C for 2.5 h. The pellet was resuspended in 500 Jll TE buffer followed by phenol-chloroform extraction and precipitation of the single stranded DNA with ethanol for 30 min. at -70 °C. The DNA pellet was washed with 70% ethanol, dried and dissolved in 200 Jll TE buffer. The uracil containing template was quantitated by analysing on an agarose gel.

Site-directed Mutagenesis

Human oocytes were washed twice with PBS containing 0.1 % BSA and then incubated with 1 :50 dilution of immune or pre-immune serum samples at RT for 30 min. Following washing with PBS (3 changes of 5 min each), the oocytes were treated with goat anti-rabbit Ig-FITC conjugate for 30 min at RT. After washing with PBS, the treated oocytes were mounted in Glyceroi:PBS (9: 1) and examined under fluorescent microscope.

Indirect Immunofluorescence on Human Oocytes

For analysis of viral DNA by dot blot, I X I o5 cells were seeded in each we11 of the 9~ well plate and infected in duplicate with 50 J.Ll of the plaque pick for I h, followed b: addition of 50 J.Ll of CM to each well. Infected cells were incubated for 5 days, afte which the culture supernatant was saved and ce11s were processed for dot blot analysi~ Ce11s were lysed with 200 J.LI of 0.5 M NaOH. The alkali was neutralized by addition o 50 J.LI of 4 M ammonium acetate. The nylon membrane was wetted in warm water an( washed in dot blot solution (1 M ammonium acetate, 0.02 N NaOH) and the cell lysatt was blotted on to the membrane using a dot blot apparatus (Bio-Rad), dried, UV eros: linked and processed for prehybridization and hybridization. For the isolation of total genomic DNA, cells infected in a 35 mm culture dish wen harvested 72 h post infection (pi) and treated with 400 J.LI of DNA extraction buffer(]( mM Tris HCI, pH 8, 0.6% SDS, 10 mM EDTA)·and 50 J.Ll of 20 mg/ml proteinase K a 37°C for 12-I6 h. The DNA was extracted twice with phenol:chloroform:isoamy alcohol (25:24: 1) and once with chloroform. For each extraction, the suspension wa! mixed by inverting the eppendorf and separated by centrifugation at 2,000 rpm for 3 mir in a microfuge. DNA was precipitated with I ml of 95% ethanol at -20°C for 4 h anc pelleted at 4,500 rpm for 20 min. The pellet was washed with 70% ethanol, dried anc resuspended in 50 J.LI of TE. DNA was digested with Hind III, resolved on a 0.8% agarose gel and processed for Southern blotting. Positive clones were amplified b) infecting cells at a multiplicity of infection (MOl) of ~1 for 10 days and the amplifiec virus. was titrated using a plaque assay. Sf9 cells were infected at -I 0 MOl fo1 expression of the r-proteins.

Screening of the Recombinant Viruses

E. coli strains deficient in specific proteases were used to study their influence on the expression of r-bZP3. BL21 (DE3) and BL21 (pLysS) deficient in ompT and ion proteases and DF5 carrying a targeted mutation of the ptr gene, were transformed with the pQE-bZP3 plasmid. Colonies obtained were grown 0/N and subcultured next morning and grown till A6oo=0.7. Cultures were then induced with 0.5 mM IPTG for 3 h. Harvested cells were checked by SDS-P AGE and immunoblotting.

Expression of r-bZP3 in Different E. coli Strains

centrifuged at 10,000 rpm for 10 min, washed with 70% ethanol and dried. DNA was resuspended in 500 J..Ll of TE containing 20 J..Lg/ml RNAase, incubated at RT for 30 min and analyzed by agarose gel electrophoresis. DNA for transfection was prepared using the Plasmid midi kit DNA purification system using protocols described in the manual.

A 1000 ml culture of cells harboring the plasmid were grown 0/N in LB Amp· Next morning the culture was chilled and cells pelleted at 4,500 rpm in a Sorvall SS34 rotor for 20 min. The supernatant was discarded and cells were washed with 100 ml of STE buffer (0.1 M NaCI, 10 mM Tris HCl and 1 mM EDT A, pH 8.0). The pellet obtained after centrifugation was resuspended in I 0 ml of GTE solution containing I mg/ml lysozyme and the mixture was incubated at RT for 20 min at 4oc. Alkaline SDS (20 ml) was added and the mixture was incubated at RT for 10 min after mixing gently by inverting the tube. Ice cold potassium acetate solution ( 15 ml) was added and the tube was chilled on ice for 15 min and then centrifuged at 18,000 rpm at 40C in a SS34 rotor. The supernatant was carefully transferred to a fresh tube, DNA was precipitated by adding 0.6 volume isopropanol and incubating at RT for 10 min and then recovered by centrifugation at 5000 rpm at RT for 30 min. DNA was rinsed with 70% ethanol, dried and dissolved in 3 ml of TE. To the nucleic acid solution 3 ml of chilled LiCI (5 M) was added, mixed and the precipitate removed after spinning at 10,000 rpm for 10 min at 40 C. DNA was precipitated from the supernatant using an equal volume of isopropanol,

Large Scale Plasmid DNA Isolation

a 1.5 ml eppendorf tube and the gel slice put into the paper cone. The tube was centrifuged for 10 minutes at room temperature, to elute the DNA into the filtrate. The filtrate was extracted with one volume of phenol I chloroform ( 1:1 vlv ) , and the DNA precipitated from the aqueous phase by the addition of 5 M NaCl to a final concentration of 1 M, and 2 - 3 volumes of ethanol at -20°C, for a few hours. The centrifuged DNA pellet was dissolved in an appropriate volume of TE.

After digestion of the plasmid DNA with appropriate restriction enzymes, the DNA fragments were resolved by electrophoresis on preparative agarose gels of a suitable percentage, and stained with ethidium bromide as described above. Depending upon the amount of DNA to be resolved on the gel, the size of the sample well varied from 1.5 em - 5 em x 0.3 em, such that the desired fragment could be cut out with a minimum of agarose accompanying it. The DNA bands were visualised under long wave UV ( 366 nm ), using a hand held monitor model UVGL-58 Mineralight Lamp, UVP, Inc., California, USA), and the desired fragment cut out as a thin agarose slice keeping the size of the slice as small as possible ) . DNA was eluted from the agarose slice by the method of Zhu et al., ( 1985). Briefly, a GeneScreen ( NEN ) or Durapore ( Millipore, GVWP 04 700 membrane was wetted with 200 ul of elution buffer ( 0.1 % SDS +50 mM Tris. HCl, pH 7.5 ), and folded over to form a cone. Meanwhile, the conical lower half of an eppendorf tube was cut off and a hole pierced in the bottom with a hot wire or needle. The membrane was placed into this cone, pushing it as far as possible. This assembly was then transferred to

Isolation of restriction fragments of DNA

The inhibition potency of our synthetic substrate, lactal (31), was determined by measuring the hydrolytic activity of ~-D-galactosidase on o-nitrophenyl ~-D­galactopyranoside.97 Each assay tube contained 2 ml of o-nitrophenyl ~-D galactopyranoside solution (500 IlM), 0.25 units of ~-D-galactosidase (source: Aspergillus oryzae; purchased from Sigma, cat. no. G-5160) and a certain concentration of lacta!. The tube was incubated at 30°C for 13 min, after which 200 III of the mixture was added to a tube containing 100 III of acetate buffer (25 mM, pH = 4.5) and 200 III of sodium carbonate solution (200 mM). The colour liberated was read spectrophotometrically at 400 nm. The control tube contained everything except the substrate, lacta!.

~-D-Galactosidase inhibition by Lactal

The Luria Bertani (LB; pH 7.5) medium was prepared in double distilled water by adding, NaCl 1%, Yeast extract 0.5%, and Tryptone 1% and sterilized by autoclaving under pressure (15 lbslinch2) for 20 min. Solid growth medium was prepared by adding 1.5% agar to LB prior to autoclaving. Appropriate antibiotics were added after cooling the medium to approximately 50-60°C. Bacterial cultures were grown in LB medium at 37°C in an orbital shaker set at 200 revolutions per minute (rpm).

Media composition and bacterial culture

Phagocytesfromtheheadkidneywerestimulatedwithphorbol12-myristate13 -acetate(PMA,SigmachemicalCo)andtherespiratoryburstwasdeterminedbytheluminol-enhancedCLmethod(ScottandKlesius,1981)

AcidphosphatasewasassayedfollowingtheprocedureofMorton(1955).Theactivityoftheenzymewasexpressedaspmolphenolformedmin'1mg'1protein.

Acidphosphatase(ACP:acidorthophosphoricphosphohydrolase)(EC3.1.3.2)

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

MCHC

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

MCV

RedBloodcellsindices

Thefishessurvivedwithoutanymortalityintheeffluentconcentration ofnominalvalues2%,5%and7%.The30,60,90and120dayschronicexposurewith20fishaddedrandomlytoeachof60by40by240cmplastictankswasbegunwithfishfromthesameoriginastheseandintheinitialacutebioassays.Flowratesmaintainedtothetanksallowedfor twovolumesturnovers24hr.

Thefisheswereexposedtodifferentconcentrationsofeffluentandthenumberoffishineachconcentrationwasrecorded.Thedataweresubjectedtoprobitanalysis(Finney,1964)andDragstedtandBehven’sequation(Carpenter,1975)todetermineLC50values.Apresumableharmlessconcentration(C)oftheeffluentwasalsocalculatedbyusingthesafefactororapplicationfactor(Sprague,1971)employingtheformulaC=Where,48hrLC50xAS24 hrLC50S= -------------------48hr LC50A=0.3(constant)Thesafeconcentrationisausefulunitofmeasurementofacceptableamountoftheeffluent,whichhasnolethalityandstresstotheanimalexposed.Approximately1/3ofLCsoofvalueofeffluentwasselectedassublethalconcentrationinthepresentstudy.Fishesweredividedinto4groupsandkeptin401glassaquariacontaining wellwaterofpH7.2.GroupI,IIandIDwerekeptin2%,5%and7%ofeffluents(Figure7)respectivelyandexposedto24,48and72(short-term)periods.Allacutelethalitytestswereconductedaccordingtothe methodsofthe AmericanPublicHealthAssociation(1985).GroupIVservedascontrol.

Bioassay

polymerase and [a-32p] UTP (specific activity 3000Ci/mmole). The Riboprobe in vitro Transcription Systems (Promega) was used to make the in vitro transcripts. According to the manufacturer's directions, 0.2-lpg of the linearized DNA template was combined with the following components, in a final volume of 20pl, at room temperature in the following order: 4pl of SX transcription buffer (200mM Tris-HCl, pH 7.5, 30mM MgCh, lOmM Spermidine, 50mM NaCl), 2pl of lOOmM DTT, 20U of RNasin Ribonuclease inhibitor, 2.5mM each of ATP, GTP and CTP (pH 7.0), 2.4pl of lOOpM UTP (pH 7.0), Spl (50pCi at lOpCi/pl) of [a-32P]UTP and 15-20U of T7 or SP6 RNA Polymerase. For carrying out cold in vitro transcription all the four nucleotides (ATP, GTP, CTP, and UTP) were added at 2.5mM concentration and the reaction volume was made up with nuclease free water. The mixture was incubated at 370C for 60 min. The reaction was stopped using the stop buffer (50mM Tris-Cl, pH 7.5, SmM EDTA, 25pg tRNA/ml) and chilled on ice. RQl RNase-free DNase was added at a concentration of lU/pg of template DNA and incubated at 370C for 15 min to remove the DNA template following transcription. The transcripts were then purified by phenol : chloroform : isoamyl alcohol and chloroform : isoamyl alcohol extractions, followed by precipitation with 2.5 volumes of absolute alcohol and 0.5 volumes of 7.5M ammonium acetate and then 0.5 volumes of 1M ammonium acetate to remove the unincorporated nucleotides. After centrifugation for 30 min at 13,000 rpm the supernatant was carefully removed. The pellet was washed with 70% ethanol, vacuum dried and dissolved in 20pl of NFW

Plasmids containing the ribozymes or substrates were linearized at their 3' end with the appropriate enzymes. The linearized DNA was purified using the Qiagen Gel Extraction kit as described before (section 7.9). In vitro transcription reaction was then carried out using both T7 or SP6 RNA

In vitro Transcription:

s promastigotes develop from procyclic to metacyclic forms, the surface LPG undergoes changes in size and carbohydrate structure. Procyclic promastigotes express a smaller LPG capable of binding to the sand-fly midgut, while metacyclic promastigotes express a larger LPG typically accompanied by changes in the terminal sugar of some of these units (McConville et al., 1992). The lectin peanut agglutinin (PNA) binds to terminal galactose of procyclic LPG but does not bind its metacyclic LPG counterparts which terminate in arabinose. This principle is used in assessing the number of metacyclics in a culture (Sacks et al., 1985). Briefly, after appropriate incubation, 107 cells were harvested and washed once with PBS followed by resuspension in 1mL PBS. PNA (1mg/ mL, prepared in PBS) was added to the cells at a final concentration of 10011g/ mL and incubated at 23°C for 1hr followed by incubation on a shaker incubator set at 40rpm for 5 min. The clumps formed were then pelleted at 100 x g for 3 min at RT. From the supernatant, 1011L was withdrawn for counting on a Neubauer's chamber to get the number of metacyclics per 107 cells

Assay for measuring the number of metacyclic parasites (Peanut Agglutination Assay)

RNA was isolated as described above. 3'RACE was carried out using a kit procured from Invitrogen (Carlsbad, CA) according to manufacturer's instructions. Briefly, 4~-tg of RNA was taken and e-DNA prepared using adaptor primer (AP) provided in the kit. This e-DNA was then used as template in a PCR reaction using a gene specific forward primer (Ld30RA3 and Ld34RA1), and the abridged universal amplification primer (AUAP) that is homologous to the adaptor primer as the reverse primer. The PCR product generated was then sequenced using T7 an SP6 primers after cloning into pGEM-TEasy vector as described below

Rapid Amplification of e-DNA ends (RACE)

x g for 10 min at RT. The supernatant was centrifuged at 1258 x g for 10 min at RT. The pellet obtained was washed 2X at 4°C in half the culture volume of Cytomix buffer (120 mM KCl, 0.15 mM CaCh, 10 mM K2HP04, 25 mM HEPES, 2 mM EDTA, and 2mM MgCh; pH 7.6) and then resuspended in chilled cytomix buffer at a density of 2 X 108 cells/mL. Electroporation: For a single electroporation, 2011g of plasmid (in water or 10mM Tris pH 8.0) for episomal expression and 5Jlg of plasmid for integration events, was added to a pre-chilled cuvette ( 4mm, BTX, San Diego, CA). 500J1L of chilled cell suspension (108 cells) processed as above was transferred to the cuvette and mixed with DNA by gently tapping and incubated on ice for 10 min. The cells were electroporated twice at 25 J.IF, 1500 V (3.75 kV /em), pausing 10 s between pulses (Robinson and Beverley, 2003) in a BioRad Gene Pulser X Cell electroporator. The cell suspension was then transferred to 5mL of mDMEM containing 20% FBS and allowed to recover for ~18hrs before antibiotic selection commenced. Selection of transformants: Selection of parasites containing recombinant DNA was carried out initially in liquid medium followed by culture with agar. After the rest period, the electroporated cells were exposed to 1011g/ mL of G418 or Hygromycin B for 48 hrs with antibiotic being increased to 2011g/ mL and 50Jlg/ mL at 48 hr. intervals. Part of the cells were then plated onto freshly poured mDMEM plates (1X mDMEM, 2% agar containing 100]lg/ mL, 500]lg/ mL or 1mg/ mL G418; or 50]lg/ mL Hygromycin B) and incubated at 23°C for 7-14 days. Individual colonies obtained on plates were cultured in liquid medium and screened appropriately. Limiting dilution was used to isolate single clones.

Preparation of Leishmania culture for electroporation: An early log phase culture of Leishmania donovani was harvested and dead cells pelleted at 129

Foreign DNA can be introduced into Leishmania cultures using electroporation. Transfected circular plasmids are maintained as episomes, while linear DNA can integrate into the genome. Preparation of DNA : DNA construct to be electroporated was generated using standard molecular biological techniques as described later. The plasmid DNA was prepared from E. coli DH5-a or XL-1 Blue cells using the EndoFree MaxiPrep kit from Qiagen (Hilden, Germany) according to manufacturer's protocol. Briefly, 200mL of overnight culture was pelleted at 6000 x gat 4°C for 15 min. The pellet was resuspended in 10mL Buffer P1 (50mM Tris-Cl, pH 8.0, 10mM EDTA, 100p.g/mL RNaseA). To this 10mL Buffer P2 (200mM NaOH, 1 %w /v SDS) was added and mixed thoroughly by vigorously inverting 4-6 times and an incubated at RT for 5 mins. Now 10mL of chilled Buffer 3 (3.0M Potassium acetate, pH 5.5) was added and mixed thoroughly by vigorously inverting 4-6 times and then the lysate was poured into a QIAfilter catridge and incubated at RT for 10 min. Subsequently a plunger was used to filter the cell lysate into a 50mL tube to which 2.5mL Buffer ER was added and inverted 10 times to mix. This was incubated on ice for 30 min. In the meantime, a QIAGEN-tip was equilibrated with 10mL Buffer QBT (750mM NaCl, 50mM MOPS, pH 7.0, 15%v /v isopropanol and 0.15%v jv Triton X-100). After incubation, the filtrate was allowed to enter the tip resin by gravity. This was followed by two washes with 30mL of Buffer QC (1.0M Nacl, 50mM Tris-Cl, and pH 7.0 and 15% v /v Isopropanol). DNA was eluted with 15mL Buffer QN (1.6M NaCl, 50mM MOPS, pH 7.0 and 15%v /v isopropanol) and precipitated by adding 10.5 mL (0.7 volumes) isopropanol at RT and centrifugation at 15,000 x g for 30 min at 4°C. The pellet was washed with 5mL endotoxin-free 70% ethanol at RT and centrifuged at 15000 x g for 10 min. The pellet obtained was air dried for 5-10 min and redissolved in a suitable volume of endotoxin-free buffer TE (10mM Tris-Cl, pH 8.0, 1mM EDTA). The concentration of the obtained DNA was estimated by measuring the absorbance at 260nm (A260) and using the known formula: DNA concentration = A260 X SOX dilution factor.

Transfection of Leishmania donovani promastigotes

For estimation of tannase activity the reaction mixture (4 ml) contained 1.0 ml of 1.0% tannic acid (prepared in citrate-phosphate buffer, pH 5.0), 2.0 ml of citrate-phosphate buffer (pH 5.0) and 1.0 ml of appropriately diluted culture supernatant. The reaction mixture was incubated at 40°C for 30 min in a water bath. The reaction was stopped by adding 4.0 ml of 2.0% BSA solution. In the control reaction, BSA was added prior to incubation. Now the tubes were left for 20 min,at room temperature, for precipitating the residual tannins and subsequently centrifuged at 10,000 rpm for 20 min. The end product, gallic acid thus formed was estimated by diluting 20 μl of the supernatant to 10 ml with DDW. Now, the absorbance at 260 nm was read against a blank (DDW) in a UV spectrophotometer (1601, Shimadzu Corporation, Japan). One unit of tannase: One tannase unit is defined as the amount of enzyme that releases 1 μmol of gallic acid from the substrate (tannic acid) per ml per min under standard assay conditions

In this method, tannase activity was estimated through spectrophotometric method by determining the concentration of the end product i.e., gallic acid, by estimating the absorbance at 260 nm. Reagents: •Tannic acid (1.0%): The solution was prepared by dissolving 1.0 g of tannic acid in 100 ml of citrate-phosphate buffer of the desired pH.•Bovine serum albumin (BSA): BSA (2.0%) was prepared in citrate phosphate buffer (pH 5.0)

Estimation of tannase activity (Deschamp et. al., 1983)

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

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

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

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

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