antibody at an appropriate dilution. The immunoreactivity was detected by enhanced chemiluminescence using an ECL detection kit (Amersham Biosciences) and were recorded on X-ray films after appropriate exposure and development. It is important to note that the blots for probing phosphorylated proteins were performed using 1% BSA as blocking agent instead ofblotto

Whole cell extracts were prepared by treating cells with lysis buffer (0.125M Tris, 4% SDS, 20% glycerol, and 10% ~-mercaptoethanol), and protein estimation was performed using CB-X protein assay kit as per manufacturer's protocol. Lysates were resolved on 12% SDS-PAGE gel, following which Western transfer was performed onto nitrocellular membranes using a BioRad Western transfer apparatus. The blots were incubated with 5% blotto (non-fat dry skimmed milk) in 0.05% PBS-Tween 20 for 1 h to block non-specific binding sites following which they were incubated for 1 h with primary antibody at an appropriate dilution prepared in 1% blotto in 0.05% PBS-Tween-20. The blots were washed 3x with 0.05% PBS-Tween-20 at 5 min intervals following which they were incubated for 1 h with secondary

SDS-PAGE and Western blot

THP-1 macrophages and human peripheral blood monocyte derived macrophages were transfected with SMARTpool Bcl-2 siRNA (15 pmol), or ER-a siRNA (100 pmol), or ER-~ siRNA (100 pmol), or with negative control siRNA (15 pmol or 100 pmol) using TranspassR2 transfection reagent. Prior to transfection, the cells were depleted of serum by washing 2x with serum-free media. The transfection complex was prepared by diluting 0.5 J!L of transfection reagent A and 1.0 J!L of transfection reagent B to 400 J!L of serum-free media and siRNA's were added to the mix at an appropriate concentration and incubated for 20 min at room temperature. The formed transfection complexes were transferred gently using a large bore pipette tip to 105 cells/well grown in 24 well plates and incubated for 6 h, following which fresh complete medium was added. Transfection efficiency was estimated by observing Cy3-fluorescence of the negative control siRNA with a Nikon TE2000E fluorescence microscope using a tetramethyl rhodamine filter (530-580 nm). For all transfections, target protein knockdown was assessed 24 h after transfection by probing extracts oftransfected cells on Western blots using appropriate antibodies

siRNA transfection

cold PBS followed by incubation with fluorophore labeled secondary antibody at appropriate dilution for 30 min at 4°C. The fluorescence was then visualized under a fluorescence microscope or analyzed by flow cytometry

Immunostaining in Live cells: Immunostaining on live cells was performed by harvesting and resuspending cells in ice-cold PBS. The cells were then incubated with an appropriate dilution of the primary antibody for 1 h at 4 °C following which two washes were given with ice-

Immunostaining in fixed cells: The cells were fixed with 4% formaldehyde for 20 min, following which two washes were given with ice-cold PBS. Permeabilization and blocking were performed simultaneously by incubating the formaldehyde fixed cells in PBS containing 0.1% saponin and 3% normal goat serum for 30 min. The cells were washed once with ice-cold PBS. The permeabilized cells were incubated with the primary antibody at an appropriate dilution for 1 h at room temperature following which three washes with ice-cold PBS was given. These cells were then incubated with fluorophore conjugated secondary antibody (IgG) for 1 hat room temperature following which three washes with ice-cold PBS were given. The nuclei were stained with Hoechst 33342 at a concentration of 1 Jlg/mL for 2 min at room temperature. The staining was then visualized under a Nikon TE2000E fluorescence microscope using appropriate filter blocks. Image acquisition was carried out using a high-resolution Retiga Exi camera (Q-imaging, Surrey, BC, Canada) and subsequent image analysis was performed on Image-Pro Plus software v5.5 (Media Cybernetics, Silver Spring, MD). Alternatively, the fluorescence staining was detected by flow-cytometry (BD-LSR, Beckton Dickinson, NJ, USA) using an air-cooled argon ion laser (488 nm) at appropriate florescence channels. Subsequent data analysis was performed on WinMdi software (Microsoft, v 2.9)

Immunocytochemistry

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

Trypan blue is a diazo vital stain which selectively colours the dead cells blue that can be visualized under light microscope. Equal volumes of cell suspension and -0.4% trypan blue dye were mixed and incubated at room temperature for 5 min. 10 J!L of stained cells were loaded on to a hemocytometer and a count of the number of viable and dead cells were made. This procedure was carried out routinely to ensure that cell viability is >95% before plating cells for experiments

Assay for cell viability by Trypan blue dye exclusion method

treatment were harvested by centrifugation at 250 x g for 5 min following which they were resuspended in 1x PBS (pH 7.5). PI was added at a final concentration of 1 J.tg/mL and incubated for 5 minutes following which the cells were pelleted by centrifugation and washed once with PBS. These cells were analyzed for uptake of PI by either flow cytometry in FL2 channel (570 nm) or by fluorescence microscopy using a G2A filter block.

Propidium iodide (PI) is a DNA intercalating fluorescent dye which is excluded by viable cells with intact membranes, however, dead and dying cells with damaged membranes take up the dye. To assess viability, cells after appropriate

Assay for cell viability by propidium iodide dye exclusion method

Biochemical and cell biology techniques

Leishmania major strain (MHOM/Su73/5ASKH) was a kind gift from Dr. Satyajit Rath, Immunobiology Laboratory, National Institute of Immunology, India. L.major promastigotes were cultured at 23°C in modified DMEM (DMEM (1 L) supplemented with sodium bicarbonate (3.7 g), HEPES (5.96 g), hemin (5 mg), biotin (1 mg), adenine (13.36 mg), xanthine (7.6 mg), triethanolamine (0.5 mL), and tween 80 (40 mg)) supplemented with 10% FCS. It is known that long term culture of L.major promastigotes results in loss of their virulence (2). Hence, to maintain the virulence of these parasites, they were propagated in mice footpad. Towards this end, the stationary phase L.major promastigotes were resuspended in Hank's balanced salt solution and 2x106 promastigotes were injected into the footpad of female BALB/c mice. 6 weeks post-infection, the infected footpad was dissected and the lesion harvested. The obtained lesion was minced and resuspended in modified DMEM supplemented with 10% FCS and placed in 23°C incubator to allow differentiation of intracellular amastigotes to promastigotes. This cycle of harvesting promastigotes from footpad lesions was performed every 6 weeks to maintain the virulent phenotype of this parasite

Protocol for propagation and maintenance of Leishmania major promastigotes

population was determined by analyzing cells immunostained with an antibody against CD14 conjugated to FITC and the purity obtained was approximately 85% monocytes, the remaining being lymphocytes. The monocytes were further cultured in the presence of human AB serum for 7 days to allow differentiation to macrophages. At the end of 7 days post-isolation, greater than 95% of cells in culture are monocytes, with the majority of lymphocytes undergoing neglect induced death.

Peripheral blood (30 mL) was collected by venipuncture from healthy male volunteers after obtaining an informed consent and in accordance to the regulations of the Institutional Human Ethics Committee (National Institute of Immunology, New Delhi, India). The peripheral blood mononuclear cell (PBMC) population was isolated by density gradient centrifugation using Histopaque 1077, where, human whole blood was layered on Histopaque 1077 and centrifuged at 400 x g for 35 min at 25°C. The mononuclear cell population was isolated from the plasma-histopaque interface, and the monocytes were further purified by washing off the non-adherent cells after incubating the total PBMC for 1 h at 3 7°C. The homogeneity

Peripheral blood monocy.te isolation and macrophage differentiation

THP-1 acute monocytic leukemia cell line (TIB-202) was purchased from American type culture collection (ATCC) (Manassas, VA). These suspension cells were maintained in culture at 37°C in RPMI-1640 medium supplemented with 10% FCS. They were sub-cultured when the cell density reached ~1X106 per mL. To induce differentiation of these monocytic suspension cell cultures to adherent macrophage phenotype, they were subjected to treatment with PMA at a concentration of 10 ng/mL for 36 h. Forty eight hours prior to experimentation, the cells were transferred to phenol-red free RPMI-1640 medium supplemented with 10% dextran-coated charcoal stripped FCS. This was performed to remove all traces of exogenous estrogens as phenol red in culture medium is known to be a weak estrogen (1) and FCS contains multiple steroid hormones which are removed upon stripping with dextran-coated charcoal. MCF-7, a breast carcinoma cell line was obtained from ATCC (Manassas, VA). They were maintained in culture at 37°C in RPMI-1640 medium supplemented with 10% FCS and were routinely sub-cultured when the cells reached a confluency of around 80%

Cell lines and cell culture

Cell culture techniques

Methods

mouse and anti-rabbit antibodies conjugated to horseradish peroxidase (HRP) were obtained from Jackson Immunoresearch (Cambridgeshire, UK). Chloroform, isopropyl alcohol, di-sodium hydrogen phosphate, sodium di-hydrogen phosphate, sodium chloride, glycine, acetic acid, hydrochloric acid, sulphuric acid, Tris, Tris-HCl, potassium chloride, di-potassium hydrogen phosphate, formaldehyde, phenol, hydrogen peroxide, and methanol were obtained from Merck (Mumbai, India). Ethanol was purchased from Fluka Chemie GmbH (Buchs, Switzerland). 17 P-Estradiol ( cyclodextrin-encapsulated), estradiol conjugated to BSA (E2-BSA), E2-BSA conjugated to FITC (E2-BSA-FITC), BSA-FITC, propidium iodide, Lipopolysaccharide from S.typhosa, Histopaque 1077, PD 98,059, Bisiondoleylmaleimide (BIM VIII), Verapamil, Pimozide, EGTA, EDTA, Phorbol myristate acetate (PMA), nigericin, amiloride, and aminoguanidine were purchased from Sigma Chemical Company (St. Louis, MO). Fluo-3acetoxymethyl ester (Fluo-3-AM), 4-amino-5-methylamino-2',7'-difluorofluorescein (DAF-FM), 5-(and -6) chloromethyl-2', 7'-dichlorodihydrofluorescein diacetate (CM-H2DCFDA), SNARF (5-(and -6)-carboxy SNARF®l-AM), Sodium Green™ tetracetate, and Hoechst 33342 were obtained from Molecular Probes (Eugene, OR). Ketamine was purchased from Neon Pharma (Mumbai, India), while Xylocaine 2% was purchased from AstraZeneca (Bangalore, India). siRNA against ER-a, ER-p, and Bcl-2 were obtained from Dharmacon (Lafayette, CO), while the Cy3-labeled negative control siRNA was purchase from Ambion (Austin, TX). The siRNA transfection reagent, TranspassR2 was procured from New England Biolabs (Ipswich, MA). The Vybrant apoptosis detection system was purchased from Pro mega (Madison, WI). Alexa fluor 488 labeled dead E. coli particles were obtained from Molecular probes (Eugene, OR). Enzyme linked immunosorbent assay (ELISA) kits for detection of IL-l p, IL-4, IL-6, IL-8, IL-12, IFN-y, and TNF were obtained from BD Biosciences (NJ, USA). All other chemicals used in this study unless otherwise mentioned were purchased from Sigma Chemical Company (St. Louis, MO).

Roswell Park Memorial Institute medium (RPMI-1640) (with and without phenol red) and Dulbecco's modified Eagle's medium (DMEM) (with and without phenol red) were purchased from Sigma Chemical Company (St. Louis, MO). Fetal calf serum and dextran-coated charcoal stripped fetal calf serum (DCC-FCS) were procured from Biological Industries (Kibbutz Beit Haemek, Israel). 0.22 J.tm membrane filters were obtained from Millipore (Billerica, MA). Deoxy-ribonucleotide (dNTP) mix, magnesium chloride (MgCh), and pGEM-TEasy sequencing vector were purchased from Promega (Madison, WI). Taq DNA polymerase was obtained from New England Biolabs (Beverly, MA), while Superscript II First strand synthesis kit and TRizol reagent were purchased from Invitrogen (Carlsbad, CA). 100 bp DNA ladder, 1 kb DNA tadder, and 6X DNA loading dye were obtained from MBI Fermentas (Ontario, Canada). Synthetic oligonucleotides were obtained from Sigma GENOSYS (Bangalore, India) or Microsynth (Germany). MinElute™ Gel extraction kit was purchased from Qiagen (GmbH, Hilden). CB-X protein assay kit was purchased from G-Biosciences (St. Louis, MO). Ammonium persulphate (APS) and N, N, N', N'-tetramethylene-diamine (TEMED) were obtained from Sigma Chemical Company (St. Louis, MO). Rainbow™ protein markers, nitrocellular membranes, and enhanced chemiluminescnence detection reagent were procured from Amersham Biosciences (Piscataway, NJ). Anti-estrogen receptor alp, anti-estrogen receptor-a, and anti-actin antibodies were purchased from Calbiochem (Darmstadt, Germany). Anti-phospho CREB, anti-total CREB, anti-phospho ERK, and anti-total ERK were obtained from StressGen Biotechnologies (Victoria, BC). Anti-Bcl-2, anti-Bax, anti-Bad, and anti-Cytochrome C antibodies were purchased from SantaCruz Biotechnology (Santa Cruz, CA). Anti-histone dimethyl lysine antibody was procured from Upstate (VA, USA). Anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was obtained from Ambion (Austin, TX). Anti-Clusterin antibody was a kind gift from Dr.C.Yan Cheng of the Population Council, New York, USA. Secondary anti-mouse antibody conjugated to Alexa fluor 488 was purchased from Molecular Probes (Eugene, OR). Secondary anti

Materials

National Institute of Immunology

Balajee S.R.

PEPTIDE LIGATION AND PROTEIN ASSEMBLAGE BY REVERSE PROTEOLYSIS

National Institute of Immunology

Nidhi Gupta

Gene Regulation by Antisense DNA and Catalytic Nucleic Acids

otal RNA was isolated from cell lines after 48 hrs of transfection using trizol (Invitrogen, U.S.A.). 32p labeled antisense HBx mRNA was in vitro transcribed using T7 RNA Polymerase and Riboprobe kit (Promega, U.S.A.), as described earlier. For generating antisense HBx probe, plasmid DNA was linearized with Bam HI and subjected to transcription. Total RNA was quantitated and equal concentration (15-20 pg) was loaded after adding loading dye (50%glycerol, 1 mM EDTA, 0.25% bromophenol blue, 0.25% xylene cyanol FF) on 1% formaldehyde-agarose gel and 1X MOPS was used as the running buffer. The gel was then run at 5 V /em length of the gel. The gel was then treated with 2.5% HCl for 15 min for depurination, 0.4N NaOH for another 15 min and then in 3 M sodium acetate for 15 min, before the transfer was set. Also the nylon membrane prior to transfer was first treated with distilled water for 5 min and then in 0.4 N NaOH for 20 min. The overnight transfer was set up using 20X sse buffer as the transfer buffer at room temperature. Thereafter, the membrane was cross-linked by uv and then dipped in 2X sse for 20 min. For pre-hybridization the membrane was soaked in Rapid hybridization buffer (Amersham Biosciences, U.K.) for 2 hr at 65oC in the hybridizing oven. The probe was then added and further incubation for 4 hrs was carried out. Post hybridization the membrane was washed thrice with 6X sse at 37oC on a shaker. The membrane was then dried on a filter paper and wrapped in a saran wrap. The membrane was then analyzed by autoradiography. For ensuring the equal loading, the formaldehyde-agarose gel was also stained with EtBr for 23s and 18s rRNA

Northern Blot Analysis

ubjected to three washes with PBST and two washes with PBS. The blot was developed using the substrate DAB (Sigma, U.S.A.) or with ECL (Amersham Biosciences, U.K.)

he protein samples were diluted with 4X sample buffer which is essentially SDS-reducing buffer (O.SM Tris-Cl, pH 6.8, Glycerol, 10% (w /v) SDS, 2-J3-mercaptoethanol, 0.05% (w /v) bromophenol blue). The samples were denatured at 1000C for 10 min and the proteins were resolved on 12-15% SDS-polyacrylamide gel at 25-30mA. For detection, the proteins were transferred on to nitrocellulose (NC) membrane (Hybond-C extra, Amersham, U.K.) at 200mA, for either 1 hr or at 12 rnA, 40C for overnight. After the transfer was over, the NC membrane was washed thrice with PBST (1X PBS with 0.1% Tween 20) and blocked with 2% BSA for 2hrs (in PBST) at room temperature. Primary antibody to HBx/Vif/ APOBEC3G-NT raised in rabbit and were diluted to 1:1,000 in PBST. One hour incubation with the primary antibody was followed by three washes with PBST (10 min each) and then 1 hr incubation with 1:1,000 dilution of the secondary antibody (Anti-rabbit IgG (Fe) HRP conjugate) was carried out. The blot was further

Westem blot analysis

After transfection the cells were harvested and protein was isolated from the celllysates. The cells from each well were pelleted at 2000 rpm for 10 min at 40C. The supernatant was carefully removed and the pellet was incubated on ice for 1 hr after adding 50pl of lysis buffer (1% triton X100, 0.1mM EDTA, 0.1mM EGTA, 1mM DTT, 1X PI, all in 1X PBS) with intermittent vortexing. The tubes were centrifuged at maximum rpm for 10 min at 40C and the supernatant, containing the proteins, was collected and stored at -700C. The purified protein fractions were quantitated using the BCA protein assay kit and the O.D. was taken at 562nm

rotein isolation from celllysate

annealing at 25°C for 5 minutes, the reaction was incubated at 42°C for one hour.

series of primers were designed to detect the levels of intact gene of interest or Rz in the cell lysate. The levels of RNA were quantitated by carrying out reverse transcriptase based-PCR using the Im.Prom-11™ Reverse Transcriptase system (Promega, U.S.A.). 1}lg of template RNA and 1}lM terminal primers were combined in 5pl reaction volume and the primer I template mix was thermally denatured at 70°C for 5 minutes and chilled on ice. A reverse transcription reaction mix of volume 15 pl was assembled on ice to contain nuclease-free water, 1X reaction buffer, 1pl reverse transcriptase, 6 mM magnesium chloride, 0.5 mM dNTPs and 1 U ribonuclease inhibitor RNasin. As a final step, the template-primer combination was added to the reaction mix on ice. Following an initia

Reverse transcri.ptase polymerase chain reaction (RT-PCR)

After transfection the cells were harvested and RNA was isolated from the celllysates using Trizol reagent (Invitrogen) and purified according to the manufacturer's directions. Briefly, the cells were lysed directly in the culture dish by adding 1ml of Trizol reagent to each well. The homogenized sample was incubated at room temperature for 5 min to permit complete dissociation of the nucleoprotein complexes. For purifying the RNA, 200pl of chloroform was added, the tubes were shaken vigorously for 15 seconds and incubated at room temperature for 2-3 min. Tubes were centrifuged at 12,000 ref for 15 min at 40C. The aqueous phase was collected, mixed with 500pl isopropanol and incubated at room temperature for 10 min. Centrifugation was carried out at 12,000 ref for 10 min at 40C. The supernatant was carefully removed and the RNA pellet was washed with 1ml of 70% ethanol by vortexing and then centrifuging at 7500 ref for 5 min. The pellet was air dried and dissolved in 20pl of NFW.

RNA isolation from celllysates

The cells were assayed for Luciferase gene expression using Luciferase Assay kit (Promega, U.S.A.). After transfection, the cells were washed twice with PBS and then lysed by adding reporter lysis buffer provided in the kit. The cell lysate was collected from individual wells in eppendorf tubes, the cells were twice freeze-thawed in liquid N2 and then centrifuged at 13,000 rpm for 10 min at 40C. The supernatant was transferred to a fresh tube. 20¢ of cell extract was mixed with lOOp! of luciferase assay reagent that was kept at room temparature. The activity was determined using a luminometer (Packard lumicount, U.S.A.

Luciferase assay

incubator until the cells were 60% confluent. For each transfection, 1-2pg of DNA was diluted in 100 pi serum free media. Also, lOpl of lipofectin reagent · was diluted in 100 pi of serum free media and allowed to stand at room temperature for 30-45 minutes. The two solutions were combined, mixed gently and incubated at room temperature for 15 minutes. The cells were washed once with 2ml of serum free medium. For each transfection, 0.8 ml of serum free medium was added to each tube containing lipofectin-DNA complexes. The complex was mixed gently and overlaid onto cells. The plate was incubated for 4-6 hrs in a CDl incubator. The medium in each well was replaced with serum containing medium and the cells were further incubated for varying periods of time at 370C. The concentration of lipofectamine 2000 was used in the ratio 1:2 or 1:3 with DNA. The Rzs and Dzs were either co-transfected with the plasmid DNA of interest or when required to be transfected alone then pBSK+/-was used as carrier plasmid for better transfection efficiency. In order to ensure uniform transfection efficiency a reporter plasmid DNA (pSV -~ gal, Promega) was used

Transfection of cell lines used was carried out u5ing lipofectin reagent (Invitrogen, U.S.A.). In a six well plate 10 s cells/ well were seeded in 2m1 medium supplemented with serum. The cells were incubated in a CD2

Materials & Methods dried. These were counted directly to determine the total counts. In duplicate tubes, 1pl of the diluted probe was added to 100pg of carrier nucleic acid (tRNA or Herring Sperm DNA) in a total volume of 100pl. To this 500pl of ice-cold 5% TCA was added, mixed thoroughly and incubated on ice for 15-20 min. Glass fiber filters were wet (in duplicate) properly with 5% TCA and then these samples were applied on to them under vacuum. The filters were washed twice with 5ml of chilled 5% TCA and then air dried after rinsing with 2m1 of acetone. All the dry filters were inserted into scintillation vials containing scintillation fluid and the counts were taken in a liquid scintillation a-counter (LKB Wallac, 1219 Rackbeta, Sweden). The percentage incorporation, specific activity and the total amount of RNA made was then calculated according to the standard procedures. % incorporation =Incorporated cpm X100 Totalcpm Total RNA made (ng) = % incorporation X 338 Specific activity of probe = Total cpm incorporated p.g of RNA synthesized Cell culture media and cell lines: All the cell lines were grown and maintained in Dulbecco' s modified Eagle's medium (DMEM) with 10% Fetal bovine serum (FBS) and 1% antibiotic-antimycotic (penicillin, streptromycin and amphotericin B). The cells were maintained at 37<>C with 5% C02 in a humidified CD2 incubator (Nuaire-IR Autoflow CD2 Water-Jacketed incubator). Transient transfection

All the cell lines were grown and maintained in Dulbecco' s modified Eagle's medium (DMEM) with 10% Fetal bovine serum (FBS) and 1% antibiotic-antimycotic (penicillin, streptromycin and amphotericin B). The cells were maintained at 37<>C with 5% C02 in a humidified CD2 incubator (Nuaire-IR Autoflow CD2 Water-Jacketed incubator)

ell culture media and cell lines

dried. These were counted directly to determine the total counts. In duplicate tubes, 1pl of the diluted probe was added to 100pg of carrier nucleic acid (tRNA or Herring Sperm DNA) in a total volume of 100pl. To this 500pl of ice-cold 5% TCA was added, mixed thoroughly and incubated on ice for 15-20 min. Glass fiber filters were wet (in duplicate) properly with 5% TCA and then these samples were applied on to them under vacuum. The filters were washed twice with 5ml of chilled 5% TCA and then air dried after rinsing with 2m1 of acetone. All the dry filters were inserted into scintillation vials containing scintillation fluid and the counts were taken in a liquid scintillation a-counter (LKB Wallac, 1219 Rackbeta, Sweden). The percentage incorporation, specific activity and the total amount of RNA made was then calculated according to the standard procedures. % incorporation =Incorporated cpm X100 Totalcpm Total RNA made (ng) = % incorporation X 338 Specific activity of probe = Total cpm incorporated p.g of RNA synthesized

To determine the percentage of incorporation and probe specific activity, 1:10 dilution of the labeled probe was made in NFW. lpl of this was spotted on to duplicate glass fiber filters (Whatman GF/ A, U.S.A.) and ai

richloro acetic acid (TCA) precipitation:

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:

with 4 ml of equilibration buffer QBT (750mM NaCl, 50mM MOPS, pH 7.0, 15% isopropanol, 0.15% Triton X-100) and the column was allowed to empty by gravity flow. The supernatant was applied to the QIAGEN-tip and allowed to enter the resin by gravity flow. The QIAGEN-tip was washed thrice with 10ml of wash buffer QC (l.OM NaCl, 50mM MOPS, pH 7.0, 15% isopropanol). The DNA was then eluted with 5 ml of elution buffer QF (1.25M NaCl, 50mM Tris-Cl, pH 8.5, 15% isopropanol). The DNA was precipitated by adding 0.7 volumes of isopropanol to the eluted DNA. It was thoroughly mixed and centrifuged immediately at 13,000 rpm for 30 min at 40C. The supernatant was carefully decanted. The DNA pellet was washed with 2 ml of 70% ethanol, and centrifuged at 13,000 rpm for 15 min at 40C. The supernatant was carefully decanted without disturbing the pellet. The pellet was air dried for 5-10 min and the DNA was dissolved in 200 p.l of RNase-DNase free water. To determine the yield, DNA concentration was determined both by Ultra Violet (UV) Spectrophotometry (DU-65 spectrophotometer, Beckman, U.S.A.) and quantitative analysis on an agarose gel using a UV Transilluminator (UVP, California, U.S.A.). All the putative clones were then screened for the correct recombinant clones by restriction enzyme digestion using appropriate enzymes. The digested samples were checked on an agarose gel along with an appropriate size marker to assess the size of the insert from the putative clones. The clones containing very small fragments were further confirmed by sequencing both strands of the DNA

For large scale plasmid DNA isolation, the bacterial cells were cultured in 100ml of LB medium with 100pg/ml of ampicillin. The cultures were grown for 8-10 hours at 37<>C with vigorous shaking (-200 rpm). Plasmid DNA was isolated using the QIAGEN Plasmid Midi kit (100). Briefly, the bacterial cells were harvested by centrifuging at 6000 rpm for 10 min at 4<>C. The bacterial pellet was resuspended in 4 ml of the resuspension buffer P1 (50mM Tris-Cl, pH 8.0, 10mM EDTA, 100l!g/ml RNase A). 4 ml of lysis buffer P2 (200mM NaOH, 1% SDS) was added, mixed gently by inverting 4-6 times and incubated at room temperature for not more than 5 min. Further 4 ml of chilled neutralization buffer P3 (3.0 M potassium acetate, pHS.S) was added, mixed gently as before and incubated on ice for 10 min. It was then centrifuged at maximum rpm for 30 min at 4<>C. The supernatant containing the plasmid DNA was immediately removed andre-centrifuged at 10,000 rpm for 15 min at 4<>C. The supernatant was now collected in fresh tubes and kept on ice. A QIAGEN-tip 100 was equilibrated

lasmid Midipreps:

added and mixed by gently inverting the tube 4-6 times. The microfuge tubes were then centrifuged at 13,000 rpm for 10 min. QIAprep spin columns were placed in 2-ml collection tubes and the supernatant was applied to these columns. These were then centrifuged for 1 min and the flow-through was discarded. The Qiaprep columns were then washed by adding 0.75ml of buffer PE and centrifuged for 1 min. The flow-through was discarded and an additional centrifugation was given for another minute to remove traces of the wash buffer. The QIAprep columns were placed in a fresh 1.5ml microfuge tube. Finally, to elute out the DNA, 50pl of buffer EB (10mM Tris-Cl, pH 8.5) or RNase-DNase free water was applied to the center of each column and then centrifuged for 1 min after letting it stand for 1 min. The flow-through contained the DNA of interest.

Each single colony (white colonies, in case of blue-white screening), was inoculated individually in Sml Luria-Bertani (LB) medium with 100p.g/ml of ampicillin. The cultures were grown for 8-10 hours at 370C with vigorous shaking (-200 rpm). Plasmid DNA was isolated using the QIAprep Spin Miniprep Kit (QIAGEN, U.K.). According to the manufacturer's directions, the pelleted bacterial cells were resuspended in 250pl of buffer P1 and transferred to a microfuge tube. 250pl of buffer P2 was then added and mixed gently by inverting the tube 4-6 times. Further, 350pl of buffer N3 was

Plasmid Minipreps:

The PCR products were ligated with T-tailed vectors (pGEM-T Easy, pTARGE-TfM from Promega or pcDNA3.1 from Invitrogen). In general, the ligation was carried out at a vector: insert molar ratio of 1:3 or 1:4 in a 10pl reaction volume finally containing 1X T4 DNA ligase buffer (300mM Tris-HCl, pH 7.8, 100mM MgCh, 100mM DTT, lOmM dATP) and lU of T4 DNA ligase (Promega, U.S.A.). The reaction mix was incubated at 160C for 16 hrs (overnight). Following the reaction, the ligated DNA was transformed into Calcium Chloride treated E.coli-DH5a or XL-Blue1 competent cells with a high transformation efficiency. The transformed cells were plated on to Luria-Bertani-Agar plates containing 100p.g/ml of ampicillin. Additionally, for blue-white screening of the colonies, 20pl of SOmg/ml of X-Gal (5-bromo-4-chloro-3-indolyl-a-D-galactopyranoside) and 10pl of 0.1M IPTG (lsopropyl-J3-thiogalactopyranoside) (Promega, U.S.A.) was used. The plated cells were incubated at 370C for 8-10 hours

Ligation and Transformation:

eppendorf tube had DNA of interest. The purified DNA fragments were checked on an agarose gel, with an appropriate marker, before setting up the ligation reaction

The plasmid DNAs for cloning, were digested with the respective enzymes, checked on an appropriate percentage of agarose gel along with 100 bp ladder or .A Hind ill marker (Promega, USA) and the required fragments were eluted from the gel using the Qiagel Gel Extraction kit (Qiagen, U.K.). According to the manufacturer's directions, the area of the gel containing the DNA fragment was excised using a clean and sharp blade, minimizing the amount of surrounding agarose excised with the fragment. The gel slice was weighed and placed in a microfuge tube. Three volumes of Gel Solubilization Buffer (QG) was added for every one volume of gel. The gel piece was then vortexed and incubated at 500C for 10 min. The contents were mixed in between, by inverting the tube few times, to ensure gel dissolution. It was then centrifuged at 13,000 rpm for 1min. The flow through in the discard column was carefully removed. Then 500 p.l of buffer PB was added to remove the traces of gel and the tube was centrifuged at 13,000 rpm for 1 min. The flow through in the discard column was removed and 750 p.l of Wash Buffer (PE) (containing ethanol) was added and the tube was centrifuged at 13,000 rpm for 1 min. The flow through was discarded and another spin at maximum for 2 min was given to remove the traces of wash buffer. The column was then put on a fresh tube and finally, to elute the DNA, 40 p.l of Tris-EDTA buffer (TE) or RNase-DNase-free water was added and then centrifuged at 13,000 rpm for 1 min. The flow through in the

Gel elution of DNA fragments:

CR conditions for amplification of vif gene of HIV -1 1. Denaturation-940C-5min 2. Denaturation-940C-30sec 3. Annealing-630C-30sec 4. Extension-720C-45sec 5. Final extension-720C-5min

The polymerase chain reaction (PCR) was carried out using the PCR Core System I (Promega, U.S.A.). 200ng of template DNA/oligonucleotide and 1 pM terminal primers were combined in 2Spl reaction volume finally containing 1X Mg free reaction buffer (500mM KCl, 100mM Tris-HCl, pH 9.0, 1.0% Triton X-100), dNTP mix with 0.2mM of each, 1.5mM MgCh and 0.62SU of Taq DNA Polymerase. 30 thermal reaction cycles from steps 2-4 were repeatedly carried out, in GeneAmp PCR 2400 machine (Perkin Elmer, USA). PCR amplification was analyzed by 1-2% agarose gel electrophoresis using a 100 bp ladder or A Hind ill marker (Promega, USA). PCR conditions for amplification of HBx gene of HBV 1. Denaturation-94oC-5min 2. Denaturation-94oC-1min 3. Annealing-42oC-2min 4. Extension-72oC-2min 5. Final extension-72oC-5min PCR conditions for amplification of hammerhead-Rz 1. Denaturation-94oC-5min 2. Denaturation-94oC-30sec 3. Annealing-42oC-1min 4. Extension-72oC-15sec 5. Final extension-72oC-2min

olymerase chain reaction

METHODS

Oligonucleotides used in this study were chemically synthesized by Sigma-Genosys (The Woodlands, TX, U.S.A.).

Oligonucleotides

DNA restriction enzymes were purchased from New England Biolabs (Massachusetts, USA) and Promega Corporation, (Madison, U.S.A.). RNase A was obtained from Qiagen (West Sussex, U.K.). DNA ligase, RNA polymerase, RNAsin, Taq DNA polymerase, T7 RNA polymerase, SP6 RNA Polymerase and alkaline phosphatase were obtained from Promega Biotech.

Enzymes

32P-a-rUTP (3000Ci/mmol) was obtained from Perkin Elmer (California, USA).

Radioisotopes

The cell lines, HEK 293 (human embryonic kidney cells) and HepG2 (human hepatocellular carcinoma) cells were obtained from ATCC. APOBEC3G-HA-293 cell line was obtained from the National Institutes of Health (Bethesda, Maryland, USA) AIDS Repository and grown according to standard procedures. Ecoli strains DHSa or XL-Blue were used for DNA cloning

ell lines and Bacterial strains

was used to amplify the oligonucleotides (Promega Biotech, Madison, USA). pGEMT-Easy and p-TARGET cloning vectors were also obtained from Promega. In vitro transcription was carried out using Riboprobe transcription system (Promega Biotech, Madison, U.S.A.). BCA protein assay kit was obtained from Pierce Biotechnology (Rockford, IL, U.S.A.). Reverse transcription was carried out using lmProm-TI™ Reverse Transcriptase kit from Promega. Luciferase activity in the cell extracts was measured using Luciferase assay System (Promega Biotech., U.S.A.).

Qiaprep spin mini kit and Qiagen plasmid midi kit (West Sussex, U.K.) were used for isolation of DNA. Isolation of DNA fragments from gel was carried out using QiaGel extraction kits or PCR products were purified using nucleotide removal kit from Qiagen (West Sussex, U.K.). PCR core system I

Kits

Agarose, ampicillin, ammonium acetate, Tris Base, EDTA, SDS, sodium-acetate, potassium-acetate, boric acid, disodium-hydrogen-phosphate, sodium-dihydrogen-phosphate, sodium chloride ethidium bromide, urea, ammonium persulphate, MOPS, glycerol, sodium bicarbonate, Triton X-100, dithiothreitol, magnesium chloride, BSA, IPTG, Orange G, DEPC, Tween-20, acrylamide, calcium chloride, trypsin, EDTA, sodium citrate, bromophenol blue, xylene cyanol FF, were obtained from Sigma-Aldrich Co. (Missouri, U.S.A.). X-gal, NTP and dNTP, sodium chloride, bis-acrylamide, TEMED, PCR buffer and Magnesium chloride for PCR, DNA markers, were from Promega Biotech Co. (Madison, U.S.A.). All other chemicals were at least of analytical grade and were from Qualigens laboratories (Bombay, India) or Merck (New Jersey, U.S.A.) Trizol reagents, DMEM, lipofectin, lipofectamine 2000, antimycotic-antibiotic, gentamicin, RNase-DNase free water were obtained from Invitrogen-GffiCO/BRL (Maryland, U.S.A.). Fetal bovine serum was obtained from Biological Industries (Beit Haemek, Israel). Luria Bertini medium and Luria Miller agar for bacterial culture were obtained from Difco Laboratories (Detroit, U.S.A.). Pre-stained rainbow protein markers, nylon and nitro-cellulose membranes, ECL reagent, all were obtained from Amersham Biosciences (Buckinghamshire, U.K.).

Chemicals

MATERIALS REQUIRED:

ATIONAL INSTITUTE OF IMMUNOLOGY

SMRITI VERMA

Cytochrome P450s in Leishmania spp.

Visible Spectrophotometer was from Shimadzu (Tokyo, Japan). 11Quant Microplate Reader was procured from Biotek Instruments Inc. (Winooski, VT). Gene Pulsar X Cell electroporator was purchased from Bio-Rad Laboratories (Hercules, CA) while the cuvettes for electroporating cells were obtained from BTX Harvard Apparatus Inc. (Holliston, MA). BD Calibur flow cytometer was purchased from BD Biosciences (San Jose, CA). Hu 13 Midi DNA gel electrophoresis apparatus was procured from Scie Plas (Cambridge, UK). Protean II and III polycacrylamide gel system and Mini Transblot cells were procured from Bio-Rad Laboratories (Hercules, CA). Electrophoresis Power supply EPS 500/400 was purchased from Pharmacia Biotech-AB (Uppsala, Sweden). Peltier Thermal Cycler -200 was purchased from MJ research (Waltham, MA). UVP Gel Doc-It System was purchased from UVP Bio Imaging Systems Inc. (Upland, CA). JEOL JEM-2100F field emission transmission electron microscope at the Advanced Instrumentation facility, Jawaharlal Nehru University was used to capture images. GC-MS was performed with an Agilent 7890A gas chromatography instrument coupled to an Agilent 5975C mass spectrometer and an Agilent ChemStation software (version G1701EA, Agilent Technologies, Palo Alto, CA) A HP-5MS capillary column (30m x 0.25mm i.d) coated with 0.25 11m film 5% phenyl methyl siloxane was used for separation

Forma II Series water jacketed C02 incubator for cell culture was purchased from Thermo Fischer Scientific Inc. (Waltham, MA), BOD incubator for Leishmania cultures was obtained from Jeiotech (Korea) while 37°C incubator for bacterial cultures was purchased from Thermotech Instruments (P) Ltd (Raipur, India). Gyratory incubation shaker (Ecotron) was purchased from Infors-HT (Bottmingen, Switzerland). Optiphot fluorescence microscope, Nikon E600W upright fluorescence microscope and Nikon confocal microscope C1 were from Nikon (Tokyo, Japan). FluoStar Optima and FluoStar Omega fluorescence readers were purchased from BMG Lab technologies Inc. (Offenburg, Germany). UV-160A UV

Instrumentation

Results are expressed as mean ±SE. An unpaired two tailed student's t-test using Sigma Plot Version 10 & 11 was used for statistical analys

Statistical analysis

epoxy resins used for infiltration and embedding are not miscible with water. This was carried out by sequentially incubating the agar blocks in 25% methanol for 5 min, 50% methanol for 7 min, 70% methanol for 10 min, 95% methanol for 20 min and finally 100% methanol for 30 min followed by two more changes of methanol for 30 min each. The blocks were then incubated with transitional solvent propylene oxide for 30 min with one change at 15 min. Epoxy resin used for infiltration penetrates the cell and fills the spaces in between providing a hard medium that can withstand the cutting and electron beams. The blocks were first incubated for 30 min with a mixture of propylene oxide and resin in a ratio of 2:1. This was followed by incubation for 60 min with propylene oxide and resin in a 1:1 ratio. Finally, the blocks were put in pure resin and kept overnight at RT under shaking conditions. The following day, the agar blocks were placed in a bean capsule and overlayed with pure resin and incubated at 55°C to allow it to harden. Sectioning and Viewing: This was carried out at the Advanced Instrumentation Research Facility at Jawaharlal Nehru University

Sample Processing: Leishmania donovani cultures to be viewed under transmission electron microscope were pelleted at 1258 x g for 5min at RT. The pellet obtained were washed with PBS (0.22p. Filtered) and then resuspended in EM Fixative ( 4% Paraformaldehyde, 25% Glutaraldehyde, 0.1M Sodium Cacodylate) which had also been passed through a 0.22 p.m filter to remove any particulate matter that may interfere later with imaging. The cells were incubated in the fixative for 4-5 hours at RT followed by overnight incubation at 4°C. Subsequently the pellets were washed with sodium cacodylate buffer (0.1M sodium cacodylate, pH 7.3). The pellet was then embedded in 3%agar to prevent loss during subsequent washings. For this, agar was added to the pellet while vortexing so that the cells and agar mix well, then the agar was allowed to set. The MCT with the agar block was then cut to extract the block which itself was cut into smaller pieces to allow the solutions that will be added later to percolate well into the agar block. Post fixation was carried out to increase contrast and stability of fine structure, by incubating with 1% osmium tetraoxide for 2hr at RT. The blocks were then washed thoroughly with distilled water. Sample dehydration has to be carried out because the

Materials and Methods Staining withER Tracker™ Blue White DPX: ER Tracker™ is a dapoxyl dye that specifically stains ER in live cells. Since the dye was not found to retain after fixation, live cell staining was performed when fixation was required before staining with antibodies against the CYP proteins. Once washed, the cells were blocked with 3% NGS prepared in 0.001% Digitonin for 30 min at 4°C. This was followed by washing cells with chilled PBS at centrifugation at 805 x g for 10 min at 4°C. Then the cells were incubated with the appropriate primary antibody prepared in 0.001% digitonin for 1h at 4°C. The unbound primary antibody was washed off with chilled PBS three times by centrifugation at 805 x g for 10 min at 4°C. Cells were then incubated with appropriate secondary antibody again prepared in 0.001 % digitonin for 1h on ice. This was followed by three washes with chilled PBS as before. ER Tracker Blue (1p.M) then added to cells which were incubated on ice for 30mins on ice. The excess dye was washed and the cells viewed under the microscope after mounting on slides with anti-fading mounting media. 3.2.C.18 Electron Microscopy

taining withER Tracker™ Blue White DPX: ER Tracker™ is a dapoxyl dye that specifically stains ER in live cells. Since the dye was not found to retain after fixation, live cell staining was performed when fixation was required before staining with antibodies against the CYP proteins. Once washed, the cells were blocked with 3% NGS prepared in 0.001% Digitonin for 30 min at 4°C. This was followed by washing cells with chilled PBS at centrifugation at 805 x g for 10 min at 4°C. Then the cells were incubated with the appropriate primary antibody prepared in 0.001% digitonin for 1h at 4°C. The unbound primary antibody was washed off with chilled PBS three times by centrifugation at 805 x g for 10 min at 4°C. Cells were then incubated with appropriate secondary antibody again prepared in 0.001 % digitonin for 1h on ice. This was followed by three washes with chilled PBS as before. ER Tracker Blue (1p.M) then added to cells which were incubated on ice for 30mins on ice. The excess dye was washed and the cells viewed under the microscope after mounting on slides with anti-fading mounting media

The protocols for immunostaining cells with different antibodies and dyes were standardized individually for the respective staining procedure: Staining with MitoTracker® Red: MitoTracker® probes diffuse passively across the plasma membrane and accumulate into the active mitochondria. 100]1M stock solutions of MitoTracker Red CMX Ros were prepared in DMSO. Cells were stained at a final concentration of 100nM for 10 min at 37°C, at RT in the dark. The excess unbound dye was washed off and the cells were ready to be viewed under the microscope or processed further for fixation and antibody staining. Staining with antibodies (Table 3.9): Log phase cells were centrifuged at 129 x g for 5 min at RT to remove all dead cells. The live cells were washed 2X with PBS to remove any adherent media and FBS. Fixation was carried out with 2% formaldehyde at RT for 15 min. The fixed cells were washed 2X with PBS by centrifugation at 805 x g at RT for 5 min. They were then blocked using 3% Normal Goat Serum (NGS) prepared in 0.01% Saponin for 30 min at RT. After a wash with PBS, the cells were incubated with appropriate dilution of primary antibody prepared in 0.01% saponin for 1h at RT. The unbound antibody was thoroughly washed off by at least three washes with PBS. Incubation with respective fluorophore conjugated secondary antibody, again diluted in 0.01% saponin, and was carried out for 45 min at RT. The unbound antibody was thoroughly washed off by at least three washes with PBS. The cells were resuspended in a small volume of PBS and mounted on a slide along with anti-fading mounting media (10% glycerol and 0.001% P-phenylenediamine)

Immunocytochemistry

injection, programmed at 20°C min-1 to 200 °C and held for 10 min, then at 10 °C min-1 to 230 °C, and finally at 5 °C min-1 to 320 °C and held for 5 min. Injection temperature was set at 260 °C. High purity helium was used as carrier gas of 1.0 mL min-1 flow-rate. The spectrophotometers were operated in electron-impact (EI) mode, full scan of 40-550 amu or selected ion monitoring (SIM) was used, the ionization energy was 70 e V. Calibration curve was generated using n-hexane stock solutions of standard ergosterol dilutions (5-300 ng/mL) in duplicates and plotting the peak area versus the concentration (Yang et al., 2009).

Gas chromatography (GC) is a common type of chromatography used in analytic chemistry for separating and analysing compounds that can be vaporised without decomposition, while Mass spectrometry (MS) is an analytical technique that measures the mass-to-charge ratio of charged particles and thus determining masses for determining the elemental composition of a sample or molecule, and for elucidating the chemical structures of molecules. Combined together the technique of GC-MS can be used to identify individual components in a mixture and also quantitate them. The sterol extracts prepared in 3.2.C.14 were dried under nitrogen (N2) gas, resuspended in n-hexane and derivatized with BSTF A (N,O-bis (trimethylsilyl) trifluoroacetamide) containing 1% TMCS (trimethylchlorosilane) at 70 °C for 1 hr. The derivative mixture was dried under N2 (gas) to remove excess BSTFA and subsequently re-dissolved in n-hexane. The column temperature was set at 100°C and held for 5 min for

GC-MS analysis of ergosterol

Chromatography is the technique of separation of compounds on the basis of their distribution/ partition between two phases. Thin Layer Chromatography (TLC) is a solid-liquid form of chromatography where the stationary phase is normally polar absorbent and the liquid phase is the mobile phase made up of a single or combination of solvents depending on the solutes to be separated. The sterol isolated by method described in 3.2.C.14, were also run on a Thin Layer Chromatogram. Standard ergosterol dilutions and samples from wild-type and half knock out parasites were spotted on a Silica Gel G plate. The sterols were resolved using a binary solvent [hexane/ ethyl acetate (75/25)]. The sterols were visualized using Mo' s stain (12.5g Ammonium molybdate (VI) tetrahydrate, 5.0g Ammonium cerium (IV) sulphate, 50mL concentrated sulphuric acid, water upto 500mL

hin Layer Chromatography of ergosterol

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

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

Spectrophotometric estimation of ergosterol

A bioluminescent ATP determination kit from Invitrogen (Carlsbad, CA) was used to quantitate ATP levels. The assay is based on luciferase' s requirement for ATP for producing light (emission maxima at 560nm). The assay was carried out as described previously (Mukherjee et al., 2002). Briefly, a standard reaction mix was prepared-1X reaction buffer, 0.1p.M DTT, O.Sp.M Luciferin and 12.5 p.g/ mL Luciferase. 100 p.L was aliquoted into each well of a 96 well white plate and a base line reading measured using Fluostar Omega using the luminometer adaptor. Then either 106 parasites or different concentrations of A TP (prepared from the stock solution provided in the kit) were added to these wells as test and standard curve samples respectively. Again luminescence was measured and the baseline subtracted from the readings. The different dilutions of ATP were used to plot the standard curve which was then used to calculate the ATP levels in cells expressed as nmol/106 cells.

Materials and Methods the MTT Lysis buffer (20% SDS , 50% Dimethyl formamide) and the O.D.s7onm was measured. The standard curve was plotted and the equation derived, used to calculate the number of metabolically viable cells in experimental groups. Percentage of viable cells was calculated by comparing the number of viable cells in treated wells with that of untreated wells. 3.2.C.13 ATP determination

he MTT Lysis buffer (20% SDS , 50% Dimethyl formamide) and the O.D.s7onm was measured. The standard curve was plotted and the equation derived, used to calculate the number of metabolically viable cells in experimental groups. Percentage of viable cells was calculated by comparing the number of viable cells in treated wells with that of untreated wells

MTT micromethod is a colorimetric assay based upon the conversion of the (yellow) MTT reagent (3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide), a tetrazole to purple formazan in the mitochondria (succinate dehydrogenase) of the living cell which is quantified by measuring the optical density at 570nm. When the amount of purple formazan produced by untreated control cells is compared to that of treated cells, the effectiveness of the agent can be deduced through the production of a dose-response curve. Parasites in their log phase were harvested and the dead cells removed at 129 x g for 5min at RT. The live cells were resuspended in phenol red-free DMEM containing 10% FBS to a cell count of 2.5 X 107. 1mL of above culture was plated into each well of a 24 well plate and appropriate treatments were given for desired duration of time. Some untreated cells were also kept aside for generating the standard curve. After appropriate treatment, from each well of the 24 well plate 200pL was aliquotted into 4 wells of a 96 well plate. To each well of the 96 well plate, 10pL of MTT solution (5mg/ mL prepared in PBS and filtered with 0.22p filter) was added and the plate incubated at 23°C for 2-3 hours till colour develops in the control cells. A standard curve was also plated by taking different dilutions of untreated cells and processed similarly. Once colour developed, the reaction was stopped by lysing the cells using

MTT assay to assess viability of Leishmania parasites

hydrolysis of the non-fluorescent derivative dichlorodihydrofluorescein. In the presence of an appropriate oxidant, dichlorodihydrofluorescein is oxidised to the highly fluorescent 2, 7 -dichlorofluorescein. Log phase cultures were taken and dead cells pelleted at 129 x g for 5 min at RT. The live cells were resuspended in fresh phenol red-free DMEM containing 10% FBS to get a cell density of 107 cells per mL. The cells were loaded with the dye (Stock solution prepared in DMSO to a final concentration of 1pg/pL) by incubating every 107 cells with 2pL of stock solution for 15 to 20 min on an end to end shaker at RT and then washed with medium. The cells were incubated for another 15 min to allow de-esterification to occur. 200pL was aliquotted into each well of a black plate and a basal reading taken at 485nm/ 520nm. Subsequently stained cells were exposed to appropriate treatments and fluorescence monitored at appropriate intervals of time. For each experiment, measurements were prepared in quadruplets and expressed as arbitrary f1uorescence intensity units (AFU)

CM-H2DCFDA (5-(and-6)-chloromethyl -21,7'-dichlorodihydro fluorescein diacetate, acetyl ester) has been used as a detector of ROS as described previously (Mukherjee et al., 2002).This probe is a non-polar, non-fluorescent dye that diffuses readily into cells, where it is trapped by

Assay for measuring intracellular ROS

Rhod-2 acetoxymethyl ester is a fluorescent long wavelength calcium indicator, where the AM ester forms are cationic, resulting in a potential driven uptake into the mitochondria making them selective detectors of mitochondrial calcium. Log phase cultures were taken and dead cells pelleted at 129 x g for 5 min at RT. The live cells were washed twice with Kreb' s buffer (118mM Sodium chloride, 5.4mM Potassium chloride, 1.2mM Magnesium chloride, 1.2mM Potassium dihydrogen phosphate, 25mM Sodium hydrogen phosphate, llmM glucose, 1.5mM Calcium chloride, pH 7.4) by centrifugation at 1258 x g for 5 min to wash off all traces of medium and FBS. Washed cells were loaded with 1:1 mixture of Rhod -2 AM (1p.g/p.L stock solution prepared in DMSO) and 20%w/v Pluronic F127 for 1h at RT in the dark with shaking. Excess dye was removed by one wash with Kreb' s buffer followed by incubation of cells at RT for a further 30min for complete hydrolysis of the dye trapped in the mitochondria. Fluorescence intensities of the stained cells were measured fluorimetrically at excitation of 530nm and emission of 576nm or alternatively acquired by flow cytometer.

Assay for measuring mitochondrial calcium

1mL medium were stained with 1p.L of the dye (1ng/mL) and incubated at 37°C for 10 min in the dark. The unbound dye was washed off with either PBS or medium and cells analysed by flow cytometry or microscopy. Nonylacridine Orange (NAO): NAO (Molecular probes) is a probe which interacts specifically with non-oxidized cardiolipin, a lipid that is exclusively localized in . the inner mitochondrial membrane (Petit et al., 1992). A stock solution of 100p.M was prepared in DMSO and 1p.l (1nM) was used to stain 107 cells in 1mL medium for 10 min at 37°C. The excess dye washed off with PBS and cells were fixed with 4% formaldehyde for 3 min. Subsequently they were analysed with flow cytometry or microscopy

Mito Tracker Green®: Mito Tracker® Green (Invitrogen, Carlsbad, CA) is an agent that interacts with mitochondrial lipids and is essentially non-fluorescent in aqueous solution, only becoming fluorescent once it accumulates in the lipid environment of the mitochondria. Since the entry of the dye is not dependent on the mitochondrial membrane potential, it can be used to measure/ compare the mitochondrial mass of cells both in live as well as fixed states. MitoTracker Green® stock solution was prepared in DMSO (1pg/pL) and stored at -20°C. To stain cells

Assay for measuring mitochondrial mass

C-1 (5,5' ,6,6' -tetrachlorol,1' ,3,3' tetraethyl benzimidazoly 1 carbocyanine iodide) is an anionic mitochondrial vital dye (10mm stock prepared in DMSO) that is lipophilic and becomes concentrated in the mitochondria in proportion to the membrane potential; more dye accumulates in mitochondria with greater potential and ATP generating capacity. The dye exists as a monomer at low concentrations that emit a green fluorescence (530nm) but at high concentrations forms J aggregates that emit red fluorescence (590nm). The ratio of the two fluorescences gives a ratiometric comparison of mitochondrial membrane potential. Following appropriate treatment, 107 (in 1mL medium) cells were transferred to an MCT containing 10pL of the working stock (0.4mM ) of the dye (final concentration of 4pM), and incubated at 37°C for exactly 10 min in the dark. This was followed by centrifugation at 1811 x g for 3 min at RT. The pellet obtained was resuspended in M199 medium containing 10% FBS and centrifuged at 1811 x g for 3 min at RT. Two more such washes were given, after which the pellet was resuspended in 2mL M199 + 10% FBS and fluorescence measured at 485nm/ 530nm and 535nm/ 590nm

Assay for measuring Mitochondrial Membrane Potential (JC-1 Staining)

were then centrifuged at 1065 x g for 5 min at 4°C. The pellet obtained was resuspended in 1mL ice cold PBS and washed 5 times with PBS. Finally the pellet was resuspended in 1mL lysis buffer (10mM Tris-HCl containing 0.1% Triton X-100) and fluorescence was measured at 380nm/525nm. To normalize different samples and account for any errors in cell number between samples, 10011L of the above sample was also used for protein estimation, carried out as described above

Monodansylcadaverine (MDC) is a selective marker for autophagic vacuoles (Biederbick et al., 1995). It is an auto-fluorescent drug accumulates in acidic compartments by ion trapping and also is thought to interact with the membrane lipids of the vacuoles. Stock MDC (50mm, prepared in acetic acid) was diluted to a concentration of 50!lM in M199 medium containing 10%FBS. 107 parasites after appropriate treatment were resuspended in 1mL of working stock and incubated in the dark for 10 min at RT. These

Assay for measuring the level of autophagy in Leishmania parasites (MDC staining)

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)

The growth pattern of wild-type and transfected cells were carried out counting the number of cell in a haemocytometer at 24hr intervals. For this equal number of cells (107 or 5 X106) of wild-type and transfected parasites in log phase were plated. An aliquot was withdrawn every 24 hours and used for counting in a counting chamber to analyse growth pattern.

Growth curve analysis

In order to create a half and complete knockouts of CYP genes, a strategy of homologous recombination was employed whereby, the allele(s) in the genomic DNA is replaced by an engineered construct, leaving the other loci unaffected. Since Leishmania is an asexual organism, a two-step targeting protocol has to be followed targeting one allele at a time. The engineered construct contains a selection marker flanked on either side by sequences of the target allele and were generated as explained in 3.2.B.17. Leishmania donovani promastigotes were electroporated with the allelic replacement constructs as described above in section 3.2.A.6. 24 hrs post electroporation, selection antibiotic (Neomycin and/ or Hygromycin, as the case may be) was added to the electroporated cultures and scaled up as described above in 3.2.A.6. Clones generated were cultured and their genomic DNAs isolated. The presence of the replacement construct integrated into the genomic DNA was checked for by PCR using primers specific for the replacement construct (Table 3.6 & 3.7). Those clones which showed the presence of the replacement construct in the genomic DNA were assayed for the level of the respective CYP protein using western blotting as described above in 3.2.B.19 and 3.2.B.20

Generation of parasites with half and complete knock-out of CYP genes

Cell surface membrane proteins were separated using a method by Snapp and Landfear (1997). 4 X 107live cells/mL were resuspended in MME buffer (10mM MPOS, 0.1mM EGTA, 1mM MgS04, 0.1%v/v Triton X-100 and protease cocktail) and incubated at 4°C for 1 hr. on an end to end rotor. This was followed by centrifugation at 3000 x g for 4°C for 10 min. The pellet obtained was washed with PBS and then lysed in 2X sample buffer and run on an SDS-PAGE followed by western blotting

Separation of cell surface proteins

Western blotting by adding 2X sample buffer as described in 3.2.B.19 and 3.2.B.20 respectively

Triton X-114 has a property of a low cloud point (23°C). At temperatures above the cloud point, detergent solutions separate into aqueous and detergent-enriched phases thus separating membrane proteins (detergent phase) from cytosolic ones (aqueous phase). Promastigotes in the logarithmic phase of growth were harvested and dead cells pelleted at 129 x g. 109 live promastigotes were resuspended into 10mL of 0.5% vI v Triton X-114 containing protease inhibitor cocktail. The cells were homogenized using a 30-40 strokes of dounce homogenizer. The homogenate was incubated on ice for 90 min with intermittent stirring. Following this insoluble material was pelleted by centrifugation at 3700 x g for 35 min at 4°C. The supernatant was incubated at 37°C for 2 hr., till layers separated well. The top aqueous layer was separated from the bottom detergent layer. Samples of both these were prepared for SD

Triton X-114 extraction of membrane proteins

For experiments, cells in the logarithmic phase were taken from slant or liquid medium and dead cells removed by centrifugation at 129 x g for 5 min at RT. The supernatant was centrifuged at 1258 x g for 10 min at RT to pellet the live cells which were then resuspended in fresh medium to a cells count of 107 cells per mL. Treatment with PAT (stock solution of 10mg/mL prepared freshly in medium) was carried out at 100, 200 and 300 Jig/mL; with miltefosine (800Jig/mL stock solution prepared in DMSO) at 10, 20, 40, 60 and 80 JIM, and with H202 at 100, 200 and 300 JIM. Ketoconazole (10mM, prepared in absolute ethanol) and clotrimazole (10mM, prepared in DMSO) were used at 10 and 30 JIM. Ergosterol (3mg/ mL prepared in chloroform or absolute ethanol) was added to culture medium at a final concentration of 5-10Jig/ mL.

Drug treatments

Cell Biology and Biochemistry Techniques

Densitometry: Densitometry was performed using Labworks™ Image Acquisition and Analysis Software, UVP Biolmaging Systems, UVP Inc. (Upland, CA).

followed by several washes with PBS-Tween (50mM PBS containing 0.1 %v /v Tween 20) to remove any traces of acetic acid. Blocking: Nonspecific sites were blocked by incubating the membrane with 3%w /v milk protein (prepared in PBS-I) at 4°C overnight. Subsequently excess blocking reagent was washed off by three washes of PBS-T, each for 15 min at RT on a shaker incubator set at 60-80rpm. Incubation with primary antibody: Appropriate primary antibody dilutions (usually 1:20,000; Table 3.8) were prepared in 0.1% PBS-T or 1% blocking reagent. Blots were incubated with primary antibody for 1hr at RT on a shaker set at 30-40rpm. Unbound primary antibody was then washed off by three washes of PBS-T, each for 15 min at RT on a shaker incubator set at 60-80rpm. Incubation with Secondary antibody : Corresponding secondary antibody dilutions (usually 1:20,000; Table 3.8) were prepared in 0.1% PBS-I or 1% blocking reagent. Blots were incubated with secondary antibody for 1hr at RT on a shaker set at 30-40rpm. Unbound secondary antibody was then washed off by three washes of PBS-T, each for 15 min at RT on a shaker incubator set at 60-SOrpm. Detection by Enhanced Chemi-Luminescence or ECL: In the presence of hydrogen peroxide, horseradish peroxidase catalyses the oxidation of cyclic diacylhydrazides such as luminal. Following oxidation, luminal is in an excited state which decays to the ground state by emitting light. ECL reagents from EZ-ECL kit (Biological industries) were used according to manufacturer's instructions. Briefly, equal volume of ECL solution A (Luminal) and B (Hydrogen peroxide) were mixed and incubated with the membrane for approximately 5 min. Excess solution was drained off and the membrane enclosed in between two sheets of transparencies taking care to remove any air bubbles trapped in between. These were then placed in an X-ray film cassette and exposed to a sheet of X-ray film under red safety light. Depending on the antibody used, exposure times varied. Subsequently the X-ray film was transferred to developer followed by fixer solutions. Excess fixer was washed off with running water and the film air-dried.

Transfer of protein onto nitrocellulose membrane: Protein resolved by PAGE were transferred onto Hybond nitrocellulose membrane (Amersham, Pharmacia Biotech, Uppsala, Sweden) at SOV for 2.5 hours at 4°C in chilled transfer buffer (192mM glycine, 25mM Tris Base pH 8.3, 20%v lv methanol). Visualisation of protein bands transferred onto nitrocellulose membrane: Following transfer, the membrane was stained with amido black (0.1 %w lv amido black in 7%v lv acetic acid) for 30-60 s at RT. To destain, the membrane was washed in excess amounts of 7% vI v acetic acid. This was

Western Blottin

and incubated at RT for 5 min. The O.D. was measured at 595nm spectrophotometrically and quantitated against a standard reference table supplied with the kit. Alternatively, a standard curve was also plotted using different concentrations of Bovine Serum Albumin (BSA) and used to quantitate protein concentration. Electrophoretic separation of proteins: Electrophoresis for protein separation was performed using the Laemmli buffer system (Laemmli, 1970) on 10 or 12% polyacrylamide gels under reducing or denaturing conditions (SDS-PAGE). Polyacrylamide gels were prepared from 30% acrylamide (30% acrylamide, 0.8% bis-acrylamide), 4X running buffer (1.5M Tris-HCl pH 8.8), 4X stacking buffer (O.SM Tris-HCl pH 6.8), 10% SDS and 10% ammonium persulfate (APS). The gels were run in tank buffer (0.025M Tris Base, 0.192M glycine and 0.1% w I v SDS) at 40-60V while samples were in stacking gel and 80-100V once they entered resolving gel. A molecular weight marker (unstained or pre-stained depending on the requirement) containing proteins of known sizes were run to evaluate the approximate molecular weights of the resolved proteins. Visualization of protein bands on Polyacrylamide gels: The protein bands resolved in the gels were stained with Coomassie Brilliant Blue R250 (0.125%w lv CBB R250, 50% v lv methanol, 10% v lv acetic acid) for 15-30 min, followed by washing off excess stain with destain (50%v lv methanol, 10%v lv acetic acid)

SDS-PAGE is used to separate proteins according to their electrophoretic mobility which is a function of the length of the polypeptide chain or molecular weight, in the presence of denaturating agents when the secondary structure is lost. One molecule of SDS binds every 2 amino acids, imparting a net negative charge that is proportional to the length of the polypeptide. When loaded on the gel matrix and placed under an electric field, the negatively charged proteins migrate towards the positively charged electrode and are separated by molecular sieve. Preparation of lysates: After appropriate treatments, cells were harvested and washed 1X in PBS to remove any traces of medium and FBS. The remaining pellet was resuspended in residual buffer. A minimal volume of 2X sample buffer (0.125M Tris HCI pH 6.8, 4% sodium dodecyl sulphate, 20%v /v glycerol, 10% P-mercaptoethanol and 0.01 % bromo-phenol blue) was added drop-wise to the pellet while vortexing to ensure complete lysis. The lysate was denatured by boiling at 99°C for 15 min and debris pelleted down by centrifugation at 10000 x g for 5 min at RT. The supernatant was decanted into a fresh tube and used immediately or stored at -70°C. Estimation of protein concentration in lysates: Total protein concentration of whole celllysates was performed using the CB-X™ Protein Assay kit, by a modification of the Bradford method (Bradford, 1976). In brief, 1mL of chilled (-20°C) precipitant solution was added to 10pL of the whole cell lysate and centrifuged at 10,000 x g for 10 min. The supernatant was discarded and the pellet resuspended in 50pL each of solubilising solution A and B. To this 1mL colour reagent was added, the samples were vortexed

Sodium Dodecyl Sulphate PolyAcrylamide Gel Electrophoresis

The DNA sequencing was carried out at the DBT sequencing facility, Department of Biochemistry, Delhi University, South Campus, New Delhi using the di-deoxy method (Sanger et al., 1977)

DNA Sequencing

between the 5' and 3' flanking regions using the sites Smai and BamHI to generate pBSK+CYP710C1Hyg

The vector pBlueScript SK+ was used as the backbone to generate the replacement constructs. Standard cloning techniques were used (Sambrook et al., 1989). CYP5122A1 allelic replacement constructs were prepared by inserting ORFs encoding Neomycin or Hygromycin resistance between 0.4 Kb 5' and 0.37 Kb 3' CYP5122A1 flanking regions cloned into the vector pBlueScript SK+, to generate the constructs pBSK+CYP5122A1Neo and pBSK+CYP5122A1Hyg respectively. The following steps were performed. (i) A 416bp 5' flanking sequence of CYP5122A1 was amplified by Hi-fidelity PCR (Table 3.6) and cloned in between unique EcoRV and EcoRI sites of pBSK+ vector. (ii) A 378bp 3' flanking region of CYP5122A1 was amplified using Hi-fidelity PCR and cloned in between BamHI and Xbal sites in pBSK+ with the 5'fragment already cloned in. (iii) ORF for Neomycin resistance was amplified from the vector pXG-GFP+2 and cloned in between the 5' and 3' flanking regions already cloned into pBSK+ vector using the EcoRI and BamHI sites, to generate the construct pBSK+CYP5122A1Neo. (iv) To generate the second replacement construct, hygromycin ORF was amplified from the vector pXG-Hyg (Kindly provided by Dr. Stephen M. Beverley, Washington University) and cloned in between the 5' and 3' flanking regions using the sites Smal and BamHI to generate pBSK+CYP5122A1Hyg. Similarly CYP710C1 allelic replacement constructs were prepared by inserting ORFs encoding Neomycin or Hygromycin resistance between 0.36 Kb 5' and 0.37 Kb 3' CYP710C1 flanking regions cloned into the vector pBlueScript SK+, to generate the constructs pBSK+CYP710C1Neo and pBSK+CYP710C1Hyg respectively. The following steps were performed. (i) A 364 bp 5' flanking sequence of CYP710C1 was amplified by Hi-fidelity PCR (Table 3.7) and cloned in between unique EcoRV and EcoRI sites of pBlueScript SK+ vector. (ii) A 378bp 3 'flanking region of CYP710C1 was amplified using Hi-fidelity PCR and cloned in between BamHI and Xbal sites in pBlueScript SK+ with the 5'fragment already cloned in. (iii) ORF for Neomycin resistance was amplified from the vector pXG-GFP+2 and cloned in between the 5' and 3'flanking region cloned into pBlueScript SK+ vector using the EcoRI and BamHI sites, to generate the construct pBSK+CYP710C1Neo. (iv) To generate the second replacement construct, hygromycin ORF was amplified from the vector pXG-Hyg and cloned in

Generation of allelic replacement constructs for generation of knock outs

Leishmania! expression vector pXG-GFP+2 was obtained as a kind gift from Dr. Stephen S. Beverley (Washington University). Full length CYP5122A1 (Ld27) had been amplified by PCR using primers (F27P3/F27P2, Table 3.4) and cloned into pGEM-T Easy vector. The ORF was then excised from pGEMT using Notl and cloned into pXG-GFP+2 vector. The transformants were selected for the insertion of the gene in the correct orientation using restriction digestion. Standard cloning techniques were used (Sambrook et al., 1989).

Generation of pXG-GFP+2-Ld27 (CYP5122Al)

thoroughly by inverting the tube 4-6 times before keeping at RT for 5 min. 4mL of chilled Buffer P3 was added and mixed immediately and thoroughly by inverting the tube 4-6 times. A cartridge was capped and the entire contents were poured into it and allowed to settle for 10 min at RT. In the meantime, a Qiagen tip was equilibrated with 20mL of buffer QBT (750mM NaCl; SOmM MOPS, pH 7.0; 15%v /v isopropanol and 0.15% triton X-100). After the 10 min incubation, using a plunger, the contents of the cartridge were transferred into the equilibrated tip and allowed to drain by gravity. The tip was then washed with lOmL of Buffer QC (1M NaCl; SOmM MOPS, pH 7.0 and 15%v /v Isopropanol). The DNA was then eluted using SmL Buffer QF (125mM NaCl; SOmM Tris-Cl, pH 8.5 and 15%v /v Isopropanol) into a corex (glass) tube by gravity flow. 3.5mL of isopropanol was added to the eluted DNA and incubated at RT for 30 min. The DNA was then precipitated at 16000 x g at 4°C for 30 min. The supernatant was discarded and the pellet was washed in 2mL 70% ethanol at 16000 x g at 4°C for 10 min. The supernatant was gently decanted; the pellet was dried to remove any traces of alcohol. Then the DNA was resuspended in ~200]lL of Buffer EB (10mM Tris-Cl, pH 8.5) provided with the kit, or alternatively with nuclease-free water. The concentration of the obtained DNA was estimated by measuring the absorbance at 260nm (A26o) and using the known formula: DNA concentration = A260 X SOX dilution factor. Purity of DNA was monitored by looking at the A26o/ A2so ratio (should be above 1.6)

Plasmid DNA was isolated in large scale using QIAprep Midiprep kit according to manufacturer's protocol. Briefly, 100mL for a high copy number plasmid and 200mL for a low copy number plasmid was cultured overnight followed by centrifugation at 4629 x g for 15 min at 4°C. The pellet was washed once with PBS and then resuspended well in 4mL Buffer P1 by vortexing. To this, 4mL of Buffer P2 was added an

MidiPrep for large scale isolation of plasmids

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

Miniprep to isolate plasmids

run on an agarose gel and band size determined by comparison against DNA ladder with bands of known sizes. Clones that were positive for the presence of gene/ plasmid were further checked by restriction digestion. For restriction digestion, plasmid was first isolated by Miniprep as described below. The digestion reactions were set according to manufacturer's protocol appropriate for the enzymes used. The products were run on an agarose gel and band size determined by comparison against DNA ladder with bands of known sizes. Clones with the desired pattern of digestion were propagated further and used

Bacterial colonies obtained by transformation were checked for the presence of plasmid or gene inserted into the plasmid by colony PCR and restriction digestion. For PCR, a master mix for all the PCR reactions to be performed was made, aliquoted into PCR vials and stored on ice. Individual colonies were picked up, numbered and streaked onto an LB agar plate followed by deposition of a few cells into the PCR mix. PCR was carried out as described above for the respective primer pairs with the exception that the initial denaturation was carried out for 7 min at 94 °C. The products were

Screening of bacterial transformants

Plasmid/Ligation mix was incubated with ultra-competent cells for 30 min on ice. This was followed by a heat shock at 42°C for exactly 45s after which the cells were chilled on ice for 2 min. lmL of LB (Luria Bertani) medium was added to the cells and incubated at 37°C in a shaker incubator for 45 min. Cells were then plated on LB agar plate containing appropriate antibiotic and/ or X-gal solution and incubated at 37°C overnight

Transformation of competent cells with DNA

Ultra competent cells were prepared by using the method described by Inoue (Inoue et al., 1990). Briefly, the DH5-a cells were grown in the SOB culture medium (20gm/L Tryptone, 5 gm/L Yeast extract, 0.5 gm/L Sodium chloride, 2.5 mM Potassium chloride and 10mM Magnesium chloride, pH 7.0) at 18°C till the O.D.6oo of 0.55 was attained. The flasks were then shifted to ice-water bath for 10 min. The cells were harvested by centrifugation at 3220 x g, all media was discarded and the cell pellet was resuspended in Inoue transformation buffer (55mM Manganese chloride, 15mM Calcium chloride, 250mM Potassium chloride, 10mM PIPES, pH 6. 7). The suspension was centrifuged at 3220 x g, the buffer discarded and cell pellet was resuspended in fresh Inoue transformation buffer. DMSO (1.5mL/20mL of buffer) was added and the cells were frozen at -70°C. Cells were checked for transformation efficiency and were used if transformation efficiency was above 5 X 108 transformed colonies / Jlg of DNA

Preparation of ultra-competent cells of E. coli DHS-a

The DNA fragments eluted from the agarose gel or purified PCR products were cloned into pGEM-T easy vector which allows efficient sequencing using the common sequencing primers T7 and SP6. SOng of the vector was used with 1J..lL of T4 DNA ligase in a 10J..lL reaction volume. The reaction was allowed to proceed at 4 °C for 16h followed by transformation into DHS-a strain of E coli following standard protocols. The transformed cells were plated onto LB-agar plates containing appropriate ampicillin (100J..lg/mL) and blue-white selection reagent (40J..lL/plate). The plate was incubated at 37°C for 12 hrs, following which white colonies were picked up for screening for the presence of the gene of interest.

Sub-cloning of PCR products into pGEM-TEasy Vecto

The PCR products generated using protocol mentioned above were purified using QIAquick PCR purification kit from Qiagen (Hilden, Germany) as per manufacturer's protocol. Briefly, S volumes of buffer PB was added to 1 volume of PCR sample. This was applied to QIAquick spin column and centrifuged at 10,000 x g for 30-60s. The flow-through was discarded and the column washed with 0.7SmL of buffer PE. After discarding the flow-through again, the column was dried by a quick spin. The DNA was eluted using 30-SO J..lL of elution buffer (Buffer EB (10mM Tris-Cl pH 8.S)) or alternatively in nuclease-free water. The concentration of the obtained DNA was estimated by measuring the absorbance at 260nm (A26o) and using the known formula: DNA concentration = A260 X SOX dilution facto

Purification of PCR produc

Germany) as per manufacturer's protocol. Briefly, the gel was solubilised by incubating it with buffer QG (composition proprietary) at S0°C for 10 min. The solubilized gel was loaded onto a binding column and centrifuged at 12000 x g for 1 min. The flow through was discarded and the column was washed once with buffer PE containing ethanol. The DNA bound to the column was eluted using the elution buffer provided with the kit, or alternatively with nuclease-free water. The concentration of the obtained DNA was estimated by measuring the absorbance at 260nm (A26o) and using the known formula: DNA concentration= A26o X SOX dilution factor.

To elute DNA from agarose gel, samples were loaded on a low-melting agarose gel. The samples were resolved and visualized under UV transilluminator, and the band of interest was excised quickly using a scalpel blade. The volume of gel slice was quantified by weighing and the DNA eluted using MinElute Gel Extraction kit from Qiagen (Hilden

Elution of DNA from agarose gel

DNA fragments were resolved on 1-2 % agarose gel containing 0.5~-tg/mL ethidium bromide in Tris-Acetate-EDTA (TAE) buffer (40mM Iris-acetate, 2mM EDTA, pH 8.1). The samples were mixed with equal volume of 2X loading dye containing bromophenol blue, and the samples resolved by applying a voltage of 5-7 V /em. The resolved DNA fragments were visualized under ultraviolet illumination (312nm) and the relative band size was determined by comparison against DNA ladder with bands of known sizes. When required the images were acquired using a UVP Gel Documentation system

Agarose gel electrophoresis

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)

get a linear amplification of serial dilutions of e-DNA. Equal quantities of RNA (2~-tg) were used for e-DNA preparation for control and treated samples. Then identical PCR reactions were set for all experimental groups, according to the primer pair. Equal volume of PCR products were visualized by agarose gel electrophoresis as described below. The bands were quantified by densitometry using Labworks™ Image Acquisition and Analysis Software (UVP Bioimaging Systems, UVP Inc., Upland, CA)

Relative expression levels of specific gene(s) in treated cells were determined by semi-quantitative PCRs. Initially standardization was carried out for each primer pair (Table 3.5) to determine cycle number to

Semi-quantitative RT -PCR

Polymerase chain reaction (PCR) was used to amplify specific nucleotide sequences from e-DNA and genomic DNA derived from L. donovani. The reactions consisted of an initial denaturation at 94°C for 5 min, followed by 20-30 cycles of denaturation at 94 °C for 30", annealing at primer specific temperature for 45s, and extension at 72°C for 30s-2 min according to product size of the primer pair. A final extension at 72°C was performed for 10 min. The PCR products were resolved on 1-2 % agarose gel containing ethidium bromide and visualized under UV illumination as described later. The specific primer pairs, their Tms and the respective product sizes are listed in Table 3.1-3.7. For High-fidelity Taq polymerase (Invitrogen, Carlsbad, CA), extension was carried out at 68°C and MgS04 was added instead of MgCh in the reaction mix in accordance to the manufacturer's instructions.

Polymerase chain reaction

1258 x g for 10 min at RT and washed 2X with PBS to remove all traces of media and FBS. D1e pellet was finally resuspended in 200p.L of resuspension solution provided in the kit. 20p.L of RNase A solution was added and incubated at RT for 2 min. 20p.L of Proteinase K and 200p.L of lysis solution were added and the cell suspension was vortexed for ~15s. This was followed by incubation at 70°C for 10 min. Pre-assembled GenElute MiniPrep Binding column was equilibrated with 500p.L of column preparation buffer by centrifugation at 12000 x g for 1min. Following incubation at 70°C, 200p.L of ethanol was added to the lysate and the vortexed for 5-10s. This lysate was then transferred to the equilibrated column and centrifuged at 6,500 x g for 1min. The column was washed 2X with 500p.L of wash buffer, by centrifugation at 6,500 x g for 3 min. To collect the genomic DNA, the column was incubated at RT with 200p.L of elution buffer for 5 min followed by centrifugation at 6,500 x g for 1 min. The genomic DNA was aliquoted and stored at -20°C for long term and 4°C for short term storage

Genomic DNA of Leishmania donovani was obtained using GenElute™ a mammalian genomic DNA Miniprep kit from Sigma Aldrich (St. Louis, MO). Briefly, 108 parasites were harvested by centrifugation at

Preparation of genomic DNA from Leishmania donovani culture

First strand synthesis of mRNA into e-DNA was performed using First strand e-DNA synthesis kit from Invitrogen (Carlsbad, CA) following manufacturer's protocol. Briefly, 4 !lg of total RNA was denatured at 65°C for 5 min in the presence of Oligo dT12-18 and dNTPs and then cooled on ice for 1 min. DTT, MgCb and RNaseOUT in lOX reverse transcriptase buffer added to the above mixture and incubated at 42°C for 2 min. 1!!L/reaction of the Superscript Reverse Transcriptase enzyme was added to the denatured RNA and incubated at 42°C for 50 min. The enzyme was denatured by heating at 70°C for 15 min. The reaction was completed with degradation of the complementary RNA strand by incubating with RNase H for 20 min at 37°C. The DNA preparation was stored at -20°C.

First strand synthesis by reverse transcription

debris, polysaccharides, and high molecular weight DNA The supernatant was gently decanted into a fresh microcentrifuge tube and 200!!L of chloroform/ mL of TRizol was added and the tube was shaken vigorously for 15s. The mixture was incubated at room temperature for 2-3 min before centrifugation at 12000 x g for 15 min at 4 °C. This resulted in the separation of the mixture into a lower organic phase and an upper aqueous phase. The aqueous phase containing the RNA was gently aspirated and transferred into a fresh microcentrifuge tube and 500!!L of isopropanol/ mL of TRizol reagent was added and incubated at RT for 10min. The mixture was centrifuged at 12000 x g for 10 min at 4 °C to isolate the RNA as a pellet. The supernatant was discarded and the pellet was washed once with 70% ethanol, centrifuged and the pellet was air-dried and re-dissolved in approximate quantity of nuclease free (DEPC-treated) water. The purity (A2so/ A260 >1.8) and concentration (A2soX dilution factor X 40) of the obtained RNA was determined by measuring the absorbance at 260nm (A26o) and 280nm (A2so). For storage, the RNA was resuspended in 1mL of absolute ethanol and stored at -70°C. Subsequently before use, the RNA was pelleted at 12000 x g for 10 min at 4°C, washed with 70% ethanol and redissolved in DEPC-treated water.

Total RNA was isolated from cells using TRizol reagent (Invitrogen, Carlsbad, CA) following the manufacturer's protocol. Briefly 2X108 cells were harvested by centrifugation at 1258 x g for 10 min, and washed 1X with PBS. The cell pellet was lysed with 2 mL ice-cold TRizol reagent. The lysate was centrifuged at 12000 x g for 10 min at 4 °C to pellet down cellular

Total RNA isolation

Molecular Biology Techniques

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

Syrian hamsters (Mesocricetus auratus), 3-6 weeks old, were used as in vivo Leishmania infection models. The clinicopathological features of the hamster model of VL closely mimic active human disease. Promastigotes in the stationary phase were harvested at 1258 x g for 10 min at RT and washed several times with sterile PBS to remove all traces of medium and FBS. They were resuspended to a cell count of 2X109 cells/mL in PBS. 10011L of this (108 parasites) were injected intra-cardially into hamsters and infection allowed to proceed for 2 months. After 2 months, the hamsters were euthanized with C02 and the spleens were harvested. The spleens were first weighed, and then cut transversely. The exposed surface was gently pressed onto a clean slide to make imprints which were allowed to air dry. Following this, the smears were fixed either in chilled methanol for 5 min or 4% formaldehyde for 10 min. The slides were washed with PBS and either stored in -70°C for later use or stained with Giemsa stain to visualize infection