PROTOCOL FOR OPTIMIZATION OF HUMIC ACID-FREE DNA FROM ALKALINE SOILS

Various strains of Escherchia coli (DH5α, XL1Blue, DH10B) were used as hosts for the propagation of recombinant vectors. In addition, Bacillus subtilis was used as a host for the expression of xylanase gene from the recombinant vector pWHMxyl. Different vectors used in this investigation are listed in

BACTERIAL STRAINS

Soil, sediment, effluent, and water samples have been collected from various hot and alkaline regions of India and Japan in sterile polyethylene bags/bottles. The samples were transported to the laboratory and preserved at 4 °C. Temperature and pH of the samples was recorded.

COLLECTION OF SAMPLES

disodium hydrogen phosphate, glacial acetic acid, glucose, glycine, iodine, magnesium sulphate, potassium chloride, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, potassium iodide, potassium nitrate, silver chloride, silver nitrate, sodium bicarbonate, sodium carbonate, sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium hydroxide, sodium acetate, sodium molybdate, sodium nitrate, potassium sodium tartarate, and sulphuric acid, yeast extract, beef extract, peptone 7. Kanamycin, ampicillin, tetracycline, chloramphenicol tryptone, urea, guanidine HCl, Luria Bertani medium, Polyvinylpolypyrrolidone (PVPP)HiMedia, India 8. Restriction enzymes, T4 DNA ligase and buffers used in cloning experiments, Taq polymerase, dNTP solution, PCR buffers, PCR grade water, 1 kb and 100 bp DNA ladders, pGEM-Teasy cloning kit New England Biolabs and Promega 9. Isoamyl alcohol, methanol, trichloroacetic acid and Tris, lysozyme, acetone, acetyl-methylcarbinol, anthrone’s reagent, Folin & Ciocalteu’s phenol reagent SRL, India 10. Wheat bran, corncob, rice straw, Prosopis juliflora, Lantana camara and sugarcane bagasse Local market, New Delhi 11. Plasmid isolation, Gel extraction and PCR cleanup kits Machery Nagel, Germany

1. Agarose, polyacrylamide, ammonium persulphate (APS), N’,N ́-Methylene-bis-acrylamide, bovine serum albumin (BSA), chloroform, β-mercaptoethanol, phenol:chloroform (1:1), protein molecular weight markers (SDS), N-bromosuccinimide (N-BS), Woodwards reagent K (W-RK), dithiothreitol (DTT), iodoacetic acid (IAA), EDTA, EGTA, amylose, amylopectin pullulan, rice starch, sodium dodecylsulphate (SDS), N',N',N',N'- tetramethyl ethylenediamine (TEMED), Xylan, 4-O-methyl-D-glucurono-D-xylan-remazol brilliant blue R (RBB- xylan), xylose, xylobiose, xylotriose, xylotetraose and xylopentaose, p-nitrophenylxylopyranoside, p-nitrophenylacetate and p-nitrophenylarabinofuranoside, oligonucleotides synthesis (primers)Sigma Aldrich Pvt. Ltd., USA 2. Dinitrosalicylic acid (DNS), malachite green sodium sulphite and Congo red, Poly activated charcoal (PAC), Central Drug House, India 4. Agar, ammonium chloride, ammonium nitrate, ammonium sulphate, citric acid, diethyl ether, diammonium hydrogen orthophosphate, ethanol, formaldehyde, glutaraldehyde, glycerol, hydrochloric acid, hydrogen peroxide, manganese chloride, perchloric acid, potassium iodide, phenol, sodium chloride, sodium citrate and sodium acetate, X-gal (5 -bromo-4-chloro-indolyl-β-D-galactopyranoside), IPTG (isopropyl-β-D-1-thiogalactopyranoside), Imidazole Merck, India 6. Acetic acid, Coomassie brilliant blue, calcium chloride, copper sulphate, dipotassium hydrogen phosphate, Qualigens, India

For synchronization, mostly ring stage parasites (10 to 12 h post-invasion) wen~ used. The parasite culture was centrifuged at 200g for 5min and the supernatant was discarded. To the pellet, 4 ml of 5% sorbitol was added, mixed gently and incubated for 15min at 37°C. The mix was shaken 2 or 3 times and centrifuged at 200g followed by washing 3 times in complete medium (list I). The culture was then maintained at 5% hematocrit in a 37°C incubator

orbitol-synchronization 0/ P./alciparum

Parasite culture was used to make a thin blood film on a glass slide. After air drying, the thin smear was fixed in methanol for about 30s. A fresh 5 to 10% , giemsa solution was prepared in phosphate buffer (list I). The slide was placed in a I staining jar and the giemsa solution was poured on the slide for 20 min and' subsequently rinsed thoroughly under running tap water. The stained parasites, were then observed under a light microscope using 100X objective.

Giemsa staining o/thin blood smear o/parasite cultures

medium. The culture volume in 75 cm2 culture flasks, was increased to 25 ml from 12 ml. The flasks were kept at 37°C and the medium was prewarmed before use. The flasks were gassed with a mixture of 5% C02, 3% 02 and 92% N2 for a i minimum of 20 seconds at a pressure of around 5 Ib/in2. The culture medium was changed daily without the addition of RBCs. Blood smears were prepared once or twice a week to check the ~tate of the cultures and the presence of gametocytes. Typically, mature gametocytes were observed after 14-17 days

Fresh stock of parasites was thawed for culture as described above. Thin blood smears were made on the fourth day after setting up the culture. When high , parasitemia with "stressed" parasites was observed, culture volume was increased by the addition of medium. At this stage, fresh RBCs were not added to the culture

Gametocyte cultures

. Jalciparum cultures were maintained as described previously (Trager and Jensen, 1976). Briefly, P. Jalciparum strain 3D7 was cultured at 37°C in RPMI " 1640 medium (list I) in 0+ RBCs supplemented with 10% AB+ human serum or : 0.5% Albumax II (complete medium). All media were preheated to 37°C and care was taken to minimize the handling time outside the 37°C incubator. Cultures were gassed with 5% CO2, 3% O2, and 92% N2 for 20 seconds and maintained at 37°C.

Maintenance of P.falciparum cultures

cryopreserved culture vial was obtained from the liquid nitrogen tank, and thawed quickly at 37°C in a water bath. To the vial, O.lv of 12% NaCl was addeq I slowly, dropwise, while shaking the tube gently. Subsequently, 10v of 1.6% NaCl I was added slowly, dropwise while swirling the tube, followed by centrifugation at 200 g at 20°C for 5 min. The supernatant was discarded and 10v of RPMI 1640 complete media was added, followed by centrifugation at 200 g at 20°C for 5 min.' After removal of the supernatant, pelleted parasites were resuspended in complete I media at 0.5% hematocrit. Cultures were gassed with 5% C02, 3% 02, and 92%' N2 and maintained at 37°C

Revival of cryo-preserved Plasmodiumfalciparum cultures

For cryopreservation of P, Jalciparum cultures, mostly ring stage parasites at a high parasitemia were obtained. The parasites were pelleted by centrifugation at 200 g for 5 min with minimum de-acceleration. To the pellet, 1.5 volume of the freezing solution (list I) was added drop-by-drop, while shaking the vial gently; the ad4ition was completed in ~ 1 min. The medium was then transferred into a sterile cryovial, which was stored in ~he liquid nitrogen tank

Cryopreservation of Plasmodiumfalciparum cultures

Human 0+ or AB+ RBC was obtained from a donor and mixed with heparin (50 units/ml of blood) and centrifuged at 500 g for 10 min with minimu1p. de-acceleration. The supernatant was removed carefully and the pelleted RBCS were washed 3 times with RPMI 1640 to remove serum and buffy coat. Equal amount of RPMI 1640 media was added to packed RBC volume to achieve 50% hem~tocrit and stored at 4°C till: further use

Preparation of RBCsfor culture

lasmodium Jalciparum strain 3D7 (MR4, American Type Culture Collection) was i used for all the experiments except where gametocyte rich culture was required. I For generating gametocytes, 3D7 A a variant of 3D7 was used. The parasite was cultured as describerl below:

lasmodiumfalciparum culture

ynthetic peptides used for various studies were custom synthesized by Peptron I (South Korea). Reagents and solution preparations have been indexed in List I

All the reagents stock and working solutions were prepared in milliQ water. The solutions were autoclaved at 121°C (15 psi pressure) for 15 minutes. Most of the , fine chemicals were purchased from Sigma (USA) unless stated otherwise. Anti-PfCDPK4 polyclonal antibody was raised in NZW rabbit, using KLH conjugate~ I 15 amino acids synthetic peptide designed from C-terminal region of PfCDPK4 kinase domain. Phosphoinositides were purchased from Calbiochem (USA)

QuikChange site directed mutagenesis kit was procured from Stratagene. The protocol used for chemical synthesis of valery 1-FT AA-CoA ( 7) is described in section 2.2.2.15.2. Solvents and chemicals used for chemical synthesis were procured from Merck and Sigma. Weinreb resin and fmoc-amino acids were procured from Novabiochem. Other materials used in this study have been listed in chapter 2

Alaterials

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

Oligonucleotides used in this study were synthesized by Rama Biotechnologies (Hyderabad, India).

Oligonucleotides

DNA restriction enzymes were purchased from New England Biolabs (Massachusetts, USA) and Life Technologies (Maryland, USA). Lysozyme and RNase A were obtained from Sigma. RNase Tl, DNA ligase, RNA polymerase, Taq DNA polymerase, lKb DNA ladder and prestained molecular weight markers for· proteins were obtained from Life Technologies (Maryland, USA). Other protein molecular weight markers were from Sigma chemical co. T4 polynucleotide kinase were purchased from Promega. T7 DNA polymerase was obtained from USB.

Enzymes and Molecular Weight Markers

L-[3,4,5-3H (N)]-Ieucine (143Cilmmol), [35S]-dATPaS, 1251-Na (350mCilml) were obtained from Amersham (England, UK).

Radioisotopes

Cancer cell lines of human origin, HUT102, T-cell leukemia; K562, erythroleukemia; COL0205; colon adenocarcinoma; MCF7, breast adenocarcinoma; A431, epidermoid carcinoma; A549, lung carcinoma and HeLa, cervical carcinoma and J774A.I, mouse monocyte-macrophage; and L929, mouse fibroblast were obtained from ATCC. All the cell Jines were maintained in RPMI 1640 supplemented with antibiotic antimycotic solution, 2 mM glutamine and I 0% heat inactivated foetal calf serum (Life Technologies, Maryland, USA). E. coli strain DH5a was used for DNA manipulation, cloning and mutagenesis. Strains CJ236 and DH5aF' were used. for oligonucleotide mediated site directed mutagenesis. BL21 (A.DE3) strain containing T7 RNA polymerase gene under the control of lac promoter, was used for expression of the recombinant proteins.

Cell Lines and Bacterial Strains

acrylamide, TEMED were obtained from Bio-Rad laboratories (California, USA). Coomassie Plus protein assay reagent was purchased from Pierce (111inois, USA). All other chemicals were at least of analytical grade and were from Qualigens . laboratories (Bombay, India). HSA was from alpha therapeutic corporation (California, USA). Bacto-tryptone, yeast extract, and bacto-agar were obtained from Difco laboratories (Detroit, USA).

Agarose, ampici11in, ammonium acetate, ammonium persulfate, 1-acetyl 2-phenyl hydrazine, ~-mercaptuahanol, boric acid, calcium chloride, chloramphenicol, citric acid, coomassie blue 0250, creatine phosphate, creatine phosphokinase, DEPC, dialysis tubing, disodium hydrogen phosphate, dithioerythritol, dithiothreitol, DMPG, DOPG, DMPA, EDT A, ethidium bromide, glucose, glycerol, GSSG, guanidine hydrochloride, heparin, haemin., HEPES, IPTG, kanamycin, L-glycine, L-arginine, lithium chloride, magnesium acetate, magnesium sulfate, MES, PEG 8000, potassium acetate, potassium chloride, RNase free BSA, SDS, sucrose, sodium acetate, sodium dihydrogen phosphate, spermidine, sodium bicarbonate, sigmacote, Tris base, Triton X-100, urea and uridine were obtained from Sigma chemical Co. (St. Louis, USA). Trizol reagent, PCR buffer, magnesium chloride solution for PCR, RPMI-1640, leucine free RPMI, DMEM, trypsin, Fetal calf serum, antibiotic-antimycotic solution were purchased from Life Technologies (Maryland, USA). NTPs, dNTPs, cation exchange resins: S-sepharose and SP-sepharose were obtained from Pharmacia Biotech (Uppsala, Sweden). Bromophenol blue, xylene cyanol, acrylamide, bis

Chemicals

Radioisotopes: dCTP[a-32p] (3000 Ci/mM), dATP[a_35s] (1250 Ci/ mM) and 35s Met (I 000 Ci/ mM), were procured from NEN Life Sciences Products, Boston, MA, USA Others: Ni-nitrilo-tri-acetic acid (NTA) affinity resin from Qiagen; Membranes for Western blotting were obtained from BioRad; Hybond N and X-ray films were from Amersham; DNA and protein analysis softwares, PCgene from IntelliGenetics, Inc., Mountain View, CA, USA and Lasergene from DNASTAR Inc., Madison, Wisconsin, USA; Ultrafilteration assembly and YM5 membranes from Amicon Corp., Lexington, MA, USA.

from Gibco BRL; ampicillin, kanamycin and neutral red were from Sigma; FCS, was obtained from Biological Industries, Hibbutz Beit, Haemek, Israel. Bacterial Strains and Plasmids: M15[pREP4] and SG13009[pREP4] from Qiagen GmbH, Hilden, Germany, DH5a, BL21 (DE3) and BL21(pLysS) from Stratagene, La Jolla, USA, DF5 cells were kindly provided by Prof. K. Dharmalingam, Madurai Kamraj University, Madurai, Tamil Nadu, India. pBluescriptll SK( +) vector from Stratagene, pQE30 from Qiagen, pBacPAK8 vector from Clonetech Laboratories Inc., Palo Alto, CA, USA and pAcSecG2T vector, from Pharmingen, San Diego, CA, USA were obtained. Kits: Poly AT® tract mRNA isolation system and Riboclone® eDNA synthesis system from Promega Inc.; Geneclean® II kit from Bio 101 Inc., La Jolla, USA;Plasmid Midi kit and QIAexpress™ from Qiagen GmbH, Hilden, Germany; Sequenase version 2.0 DNA sequencing kit and Multiprime DNA labeling system from Amersham, Little Chalfont, Buckinghamshire, UK; BacPAK™ baculovirus expression system from Clonetech and Baculogold™ transfection kit from Pharmingen. Primers: Various oligonucleotide primers used were custom made by Rama Biotechnologies India Pvt. Ltd., Secunderab_ad, AP, India. Enzymes: Various restriction enzymes used and Vent DNA polymerase were procured from New England Biolabs, Beverly, MA, USA. Taq DNA polymerase was obtained from Stratagene. Antibodies and Conjugates: Goat anti-GST Ab was obtained from Pharmacia Biotech, Uppsala, Sweden. The following secondary revealing Abs were used: i) goat anti-mouse immunoglobulin G (lgG)-horse radish peroxidase (HRPO) from BioRad; ii) goat anti-rabbit IgG-HRPO (Pierce Chemical Co.); iii) goat anti-monkey IgG-HRPO (Sigma); iv) anti-rabbit-FITC and v) anti-goat-HRPO (Reagent Bank, Nil, New Delhi) vi) anti-mouse FITC (Dakopatts a/s, Glostrup, Denmark).

Chemicals: Tris, glycine, acrylamide, N', N'-methylene bisacrylamide, sodium dodecyl sulfate (SDS), N', N', N', N'-tetramethylethylene diamine (TEMED), ammonium persulfate (APS), ~-mercaptoethanol (BME), 4-chloronaphthol, urea, guanidine HCl, guanidine isothiocyanate (GITC), sarcosyl, sodium citrate, phenol, ficoll, polyvinylpyrrolidone (PVP), agarose, bromophenol blue, Coomassie brilliant blue, ethidium bromide, calcium chloride and bicinchoninic acid (BCA) were obtained from Sigma Chemical Co., St. Louis, MO, USA. LMP agarose and isopropyl ~-D thiogalactopyranoside (IPTG) were from Amresco, Solon, USA. Molecular weight standards were obtained from Gibco-BRL, Grand Island, NY, USA, or Bio-Rad Laboratories, Hercules, CA, USA. Reagents for enzyme immunoassays viz., bovine serum albumin (BSA), orthophenylene diamine (OPD) were procured from Sigma, while Tween-20 was obtained from Amresco. Reagents for conjugation and immunization, viz. diphtheria toxoid (DT) and tetanus toxoid (TT) were from Serum Institute, Pune, India while glutaraldehyde, L-lysine, 2, 6, 10, 15, 19, 23-hexamethyl-2, 6, 10, 14, 18, 22-tetracosa-hexane (Squalene) and mannide monooleate (Arlacel A) were procured from Sigma; Pergonal® was obtained from Laboratoires Serono S.A., Aubonne, Switzerland; Sodium phthalyl derivative of lipopolysaccharide (SPLPS) was kindly provided by the lmmunoendocrinology Laboratory, National Institute of Immunology (Nil), New Delhi. Reagents used in the estimation of progesterone such as gelatin, charcoal, dextran, 3H-progesterone and anti-progesterone Ab were provided by the WHO Matched Reagent Assay Programme while diphenoxazole (PPO), 1-4 bis (5-phenyl-2-oxazolyl) benzene (POPOP), and mercury-[(o-carboxyphenyl)thio]ethyl sodium salt (Thimerosal) were obtained from Sigma. Media and Antibiotics: Bacto-tryptone, bacto yeast extract and bacto agar were from Difco Laboratories, Detroit, USA; Grace's insect cell medium and antibiotic-antimycotic

REAGENTS

All computer software facilities were provided by the NII computer centre.

Computer software.

Nitrocellulose membranes ( BA85 ) were obtained from Schleicher and Schuell, Germany. GeneScreen and GeneScreen Plus membranes were from DuPont, USA. Millipore membranes ( 0.45 um ) were from Millipore Corporation, USA. 3 MM and 1 MM chromatography filter papers were from Whatman Ltd, U.K.

other Materials.

All antisera were obtained from the reagent bank at National Institute of Immunology, New Delhi.

Antisera.

Intensifying screens were from Kiran X-ray Screens, India. X -ray films were from Agfa -Gevaert, Belgium, Kodak, USA, or Hindustan Photo Films, India. Developer and fixer were from Hindustan Photo Films, India.

Materials for autoradiography.

32p -dCTP specific activity 400 or 800 Ci 1 mmole was from Amersham, UK or from New England Nuclear division of DuPont, USA. 125I was from Amersham.

Radioactive chemicals.

Ampicillin, tetracycline, chloramphenicol and gentamycin were from Sigma. Geniticin ( G418 ) was from Gibco Laboratories, USA.

Antibiotics.

Restriction endonucleases, T 4 DNA 1 igase, DNA polymerase I large fragment Klenow ) , bacterial alkaline phosphatase BAP were from BRL, USA and New England Biolabs, USA. Lysozyme and RNase A were from Sigma. Thermus aquaticus thermostable DNA polymerase was kindly provided by Cetus Corporation, California, USA.

Enzymes.

GTG ) were from FMC Bio Products, USA. SDS, ethidium bromide, calf thymus DNA, cesium chloride, tris base, dithiothreitol, IPTG, X -gal, DAB, ficoll, PVP, chloroquine, coomassie brilliant blue, amido black, bovine serum albumin, were from sigma Chemicals Company ( Sigma ) , USA. DEAE -dextran was from Pharmacia, Sweden. Nick translation kits were from Bethesda Research Laboratories BRL ) , USA, and Amersham International plc, UK. Lipofectin was kindly provided by Syntex, Inc. , USA. J3hCG RIA kit was from ICN Micromedic Systems, Inc., USA. Purified hCG 13,000 I.U./ mg was kindly provided by Dr. Y.Y. Tsang, Population Council, USA. HBsAg detection kit was from Abbott Laboratories, USA. Protein molecular mass standards were from Bio Rad Laboratories, USA. DNA size markers were from BRL. All other chemicals were from Glaxo Laboratories, India, and E. Merck, India.

Acrylamide, bisacrylamide, ammonium persulphate, Bio -gel P-4 and TEMED, were from Bio -Rad Laboratories, USA. Agarose ( SeaKem ) and low gelling agarose ( NuSieve

Chemicals.

Bacto -tryptone, Bacto -agar and Bacto -yeast extract were from Difco Laboratories, Detroit, USA. Fetal calf I serum, Ham s F-12 medium ( DMEM ) , I Iscove s Laboratories, USA.

Media.

The bacterial strains used in this study were ~.coli Kl2 strains, HBlOl ( F-, hsd S20 ( rB-, mB-) , supE44, ara14, ~-, galK2, lacYl, proA2, rpsL20, xyl-5, mtl-1, recA13 ] (Boyer et al., 1969 ), and JM105 ( thi, rpsL, endA, sbcB15, hsdR4, ( lac-proAB ), {F1, traD36, proAB, laciqZ M15 } ]. The mammalian cell lines used are listed in Table 1. Rat-2 is an established rat fibroblast cell line. FWIL (Larrick et al., unpublished) is a human myeloma cell line derived from the fusion of U266 IgE myeloma cells with WIL-2 lymphoblastoid cells. Rat - 2 and FWIL cell lines were kindly provided by Dr. J.W. Larrick, Cetus Corporation, USA. The other four cell lines were obtained from American Type Culture Collection ( ATCC ). The plasmids used in this study are described in Table 2.

Bacterial strains, cell lines and plasrnids.

Franklin Lakes, NJ, USA. Glass micro-slides and coverslips were purchased from Blue Star, Polar Industries and Co., India.

immunoglobulins-FITC conjugates were obtained from Pierce, Rockford, IL, USA. Anti-. rabies monoclonal antibody conjugated to FITC was purchased from Centocor Inc, Malvern, P A, USA. Others: Sodium chloride was purchased from S. D. fine-chem. Ltd., Mumbai, India, Lipofectamine and Trizol were obtained from Gibco-BRL, Rockville, MD, USA. UM-449 cell line was a kind gift by Prof. Nirbhay Kumar (TheW. Harry Feinstone Department of Molecular Microbiology and Immunology, John Hopkins University-School of Public Health, N. Wolfe Street, Baltimore, MD 21205, USA), COS-7 cell line was kindly provided by Dr. Chetan Chitnis (Malaria Research Group, International Center for Genetic Engineering and Biotechnology, New Delhi, India), MNA cell line, CVS-11 rabies virus and Standard Rabies Immune Globulin were kindly gifted by Dr. Charles E. Rupprecht (Rabies section, Division of Viral and Rickettsial Diseases, Centres for Disease Control and Prevention, Atlanta, Georgia 30333, USA), Nickel-nitrilotriacetic acid (Ni-NTA) was procured from QIAGEN, ultrafiltration assembly and YM30 membrane from Amicon Corp., Lexington, MA, USA. Bicinchoninic acid (BCA) was purchased from Pierce. Complete and incomplete Freund's adjuvants were procured from Difco laboratories. Nitrocellulose membrane, Tefzel tubing, gold microcarriers and Helios gene gun assembly were obtained from Bio-Rad. T -25, T -75 tissue culture flasks, 6-well tissue culture plates, 8-well tissue culture chamber slide and 96-well microtitration plates were procured from Nunc a/s, Rosakilde, Denmark. The 24-well tissue culture plates were procured from Corning glass works, Corning, NY, USA. The 96-well tissue culture plates were purchased from Becton Dickinson and Co.,

Bacterial strains and plasmids: DH5a and BL2l(DE3)pLysS strains of E. coli were purchased from Stratagene, La Jolla, CA, USA. SG13009[pREP4] E. coli strain was obtained from QIAGEN GmbH, Hilden, Germany. Expression vector, VRI 020 was a kind gift from VICAL Incorp., San Diego, CA, USA, pKB3-JE-13 clone was procured from ATCC, Rockville, MD, USA, pQE30 vector was procured from QIAGEN and pRSET-A vector was acquired from Invitrogen Corp., Carlsbad, California, USA. Kits: PCR-Script™ Amp cloning kit was obtained from Stratagene. Plasmid DNA purification mega kit was purchased from QIAGEN. The pGEM-T Easy cloning kit was purchased from Promega, Madison, WI, USA. Primers and Enzymes: Various oligonucleotide primers were custom made by Rama Biotechnologies, India Pvt. Ltd., Secundrabad, AP, India, Sigma-Genosys Ltd, New Delhi, India and Microsynth GmbH, Hilden, Germany. Restriction enzymes were obtained from New England BioLabs (NEB), Beverly, MA, USA and Promega, Taq DNA polymerase and T4 DNA ligase were bought from Promega. Shrimp Alkaline Phosphatase was bought from Amersham. Molecular weight markers: pGEM DNA markers were procured from Promega, /..DNA-Hind III digest and 1 kb DNA ladder were purchased from NEB. Prestained SDS-PAGE standards were obtained from Bio-Rad, Hercules, CA, USA. Antibodies and Conjugates: Rabbit anti-mouse IgG (whole molecule) conjugated to horseradish peroxidase (HRPO) was procured from Dako A/S, Glostrup, Denmark. Goat anti-mouse IgG-FITC conjugate was procured from Sigma. Mouse monoclonal antibody isotyping kit was also purchased from Sigma. Goat anti-mouse immunoglobulins-HRPO, goat anti-rabbit immunoglobulins-HRPO, rabbit anti-goat IgG-HRPO and goat anti-rabbit

Chemicals: Tris, glycine, acrylamide, N, N'-Methylene-bisacrylamide, sodium dodecyl sulfate (SDS), P-mercaptoethanol, N, N, N', N'-Tetramethylethylenediamine (TEMED), phenol, ethidium bromide, ethylenediaminetetraacetic acid (EDTA), agarose, Bromophenol blue, Coomassie brilliant blue-R250, calcium chloride, sodium acetate, glucose, lysozyme, glycerol, chloroform, lithium chloride, phenylmethyl sulphonyl fluoride (PMSF), spermidine, saponin, paraformaldehyde were procured from Sigma Chemical Co., St. Louis, MO, USA. Low melting point (LMP) agarose, isoamyl alcohol, sodium deoxycholate, oxidized glutathione, reduced glutathione, dimethyl sulfoxide (DMSO), 4-chloro-1-naphthol, isopropyl-P-D-thiogalactopyranoside (IPTG) and 5-bromo-4-chloro-3-indolyl-P-D-galactopyranoside (X-gal), were purchased from Amresco, Solon, Ohio, USA. Ammonium persulfate and guanidine hydrochloride were purchased from Amersham, Cleveland, Ohio, USA. Alum was procured from Superfos Biosector, Elsenbakken, Frederikssund, Denmark. Reagents for Enzyme Immunoassay: Bovine serum albumin (BSA) and orthophenylene diamine (OPD) were purchased from Sigma. Tween-20 (polyoxyethylene-20-sorbitan monolaurate) was obtained from Amresco. Media and Antibiotics: Bacto tryptone, bacto yeast extract and bacto agar were obtained from Difco laboratories, Detroit, USA, Dulbecco's Modified Eagle's Medium (DMEM) and RPMI-1640 media were purchased from Sigma. Antibiotics such as gentamycin sulfate, ampicillin (sodium salt) and kanamycin were also obtained from Sigma. Fetal calf serum (FCS) was procured from Biological Industries, Hibbutz Beit, Haemek, Israel.

REAGENTS

Throughoutthepresentwork,unlessotherwisegiven,thecontrolandexperimentalvaluesarethereplicatesofatleast6independentobservations.Thedataareexpressedasx±SE.StatisticalcomparisonsweremadebyStudents‘t’test(SnedecorandCochran,1967).Whereverneeded,estimatesofdegreesofstatisticalsignificantdifferencesbetweengroupsweremadebyanalysisofvariance.Allregressioncalculationsusedstandardprogrammesforlinearleastsquareregressionandcomparisonoftheslopesoftheregressionlineswereprepared.Correlationanalysiswasusedtorecordanyinteractionbetweentheparametersunderscrutinyandbodyweightofthefish.ThegeneralequationY-awborlogy=loga+blogwasusedtoshowvariousallometricrelationshipsbetweenbodyweightandO2uptakerate.WherevernecessarythedatawasalsosubjectedtocomputeranalysisusingaCompaq1702computerdevelopedbyWIPRO,Bangalore,India.

Statistics

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:

Ethylene glycol tetraacetic acids (EGTA), Ethylene diamine tetraacetic acid (EDTA), Dimethyl Sulfoxide (DMSO), Dimethyl formamide (DMF) and Triton-X 114 were obtained from Sigma Chemical Company (St. Louis, MO). Heptane, hexane, chloroform, methanol and ethyl acetate (HPLC grade) were obtained from Merck Specialities Pvt. Ltd. (Mumbai, India). All chemicals were of the highest reagent grade. Other chemicals, unless specified, were from Sigma Chemical Company (St. Louis, MO). All plastic-wares were purchased from Nunc (Denmark), BD Falcon (Franklin Lakes, NJ), Axygen (Union City, CA) or Griener Bio-One (Frickenhausen, Germany

Iris, Acrylamide, N, N'-methylene-bis-acrylamide, Ammonium persulphate (APS), N,N,N' ,N' ,-tetramethylene-diamine (TEMED), and Coomassie Blue R-250 were obtained from Sigma Chemical Company (St. Louis, MO). CB-XlM Protein Assay kit was purchased from G-Biosciences (Maryland Heights, MO). RainbowlM protein molecular weight markers were obtained from Amersham Pharmacia Biotech (Uppsala, Sweden). Amersham LMWlM Calibration kit for SDS electrophoresis, Amersham ECL™ DualValue Western Blotting Marker was obtained from GE Healthcare (Buckinghamshire, UK). Enhanced chemiluminescence (ECL) components (Hypercassette Blue, Hyper-film ECL) were obtained from Amersham Biosciences (Piscataway, NJ). EZ-Chemiluminescence kit was obtained from Biological Industries (Kibbutz Beit Haemek, Isreal). Antibodies against CYP5122A1 and CYP710C1 were raised in rabbits against peptides specific for the proteins by Abexome Bioscience (Bangalore, India). Horse Radish Peroxidase (HPR) -conjugated goat anti-rabbit IgG and goat anti rat IgG were obtained from Jackson Immuno Research Laboratories Inc. (West Grove, PA USA). Alexa Flour® 488 and Alexa Fluor® 594 conjugated goat anti-rabbit IgG were obtained from Invitrogen (Carlsbad, CA). Rabbit anti-Tubulin-a and anti-Tubulin-p antibodies were obtained from Neomarker (Fremont, CA) Anti-BIP antibody was obtained as a kind gift from Dr. J. D. Bangs, University of Wisconsin (Madison, USA). Mouse monoclonal IgG anti-GAPDH antibody was from Santa Cruz Biotechnology Inc., CA. Normal Goat Serum (NGS) was procured from GIBCO BRL (Gaithersburg, MD). MitoTracker® Red, MitoTracker® Green FM, ERTracker™ Blue White DPX, JC-1 (5S,6,6'-tetrachloro-1X3,3'-tetraethylbenzimidazolyl carbocyanine iodide), Rhod-2,AM, CM-H2DCFDA (5-(and-6) chloromethyl-2' ,7' -dichlorodihydrofluoroscein diacetate, acetyl ester), Syto 11 green nucleic acid stain, Pluronic F127, Hoechst, ATP determination kit, Nonyl Aridine Orange (NAO) were obtained from Molecular Probes (Eugene, OR). Propidium iodides, Giemsa stain, Peanut agglutinin, Monodansyl Cadaverine, MIT reagent (Thiozolyl blue tetrazolium bromide) were obtained from Sigma Aldrich (StLouis, MO).

Dulbecco' s Modified Eagle's Medium (DMEM), Roswell Park Memorial Institute medium (RPMI-1640), Phenol red free DMEM, and Medium 199 (M199) were purchased from Sigma Aldrich (St. Louis, MO). Gentamycin was procured from GIBCO BRL (Gaithersburg, MD). Foetal Bovine Serum (FBS) was purchased from Biological Industries Ltd. (Kibbutz Beit Haemek, Isreal). Brain Heart Infusion Agar was obtained from Pronadisa (CONDA Laboratories, Madrid, Spain). LB agar was obtained from Invitrogen Technologies (Carlsbad, CA) while LB broth was obtained from Himedia (India). Sodium bicarbonate, HEPES buffer, Hemin, Biotin, Glucose, Adenine, Xanthine, Tween 80, DMSO and Triethanolamine were obtained from Sigma Aldrich (St Louis, MO). Potassium Antimony Tartrate (PAT), Kanamycin, Ampicillin, and Ergosterol were obtained from Sigma Aldrich (StLouis, MO). Miltefosine was obtained from Cayman Chemicals (Ann Arbor, MI). Hydrogen Peroxide (H202) was obtained from Merck Specialities Pvt. Ltd. (Mumbai, India). Hygromycin B was obtained from Sigma Aldrich (St. Louis, MO) and Invitrogen (Carlsbad, CA). Deoxy-ribonucleotide ( dNTP) mix, Magnesium chloride (MgCh), and pGEM-TEasy vector were obtained from Promega (Madison, WI). Taq DNA Polymerase and all restriction enzymes used were procured from New England BioLabs (Beverley, MA). 1 Kb DNA Ladder, 6X Gel Loading dye were obtained from MBI Fermentas (Ontario, Canada). Synthetic oligo-nucleotides were obtained from Sigma GENOSYS (Bangalore, India) and Microsynth (Germany). Platinum High Fidelity Taq Polymerase (HiFi Taq), Superscript Reverse Transcriptase, oligo dT12-1s, Dithiothretol (DTT), RNase OUTfM, RNase Hand Trizol® were procured from Invitrogen Technologies (Carlsbad, CA). DEPC, Agarose, Calcium chloride (CaCh), Blue White Select Screening Reagent, Proteinase K, Ethidium bromide, were obtained from Sigma Aldrich (St. Louis, MO). Low melting agarose (ultrapure grade) was procured from United States ~iochemical (Cleveland, OH). Plasmid MiniPrep kit, MidiPrep kit and EndoFree Maxiprep kit, PCR Purification kit and Gel Extraction kit were obtained from Qiagen (GmbH, Hilden). GenElute™ Mammalian Genomic DNA MiniPrep kit was obtained from Sigma Aldrich (St. Louis, MO)

MATERIALS

3.0–5.0, phosphate buffer for pH 6.0–8.0 and Tris-HCl buffer for pH 9.0) were used. •pH stability: The pH stability of the selected tannases was examined in the range of 3.0–9.0 by incubating the enzyme samples for 6 h in different buffers. Tannase activity was estimated under standard assay conditions. •Temperature tolerance: Temperature tolerance of the tannases was examined by assaying their activity at different temperatures in the range of 20 to 80ºC. •Temperature stability: Temperature stability of the tannases was determined by incubating them in the temperature range of 20 to 70 ºC for 6 h. After the incubation tannase activity (%) was determined under standard assay conditions. •Organic solvent stability: In order to determine the suitability of the selected tannases for organic synthesis, their stability was determined in different organic solvents. Experimentally, 10 mg of each of the crude lyophilized tannase from the selected cultures were mixed with 1.0 ml of the following organic solvent: a) Hexane b) Methanol c) Propanol d) Isoamyl alcohol e) Petroleum ether f ) Chloroform The mixture was incubated for 6 h at optimal temperature and the organic solvents were then decanted and the residues were dried in a vacuum desiccator. These dried samples were dissolved in 1.0 ml of citrate phosphate buffer (50 mM, pH 5.0) and the tannase activity was determined under standard assay conditions. The tannase activity thus obtained from each culture were compared with initial tannase activity. Finally, on the basis of tannase titres produced per ml and desirable biochemical properties, the best tannase producer was selected for further investigations

The procedure of Sharma et al. (2000) was used to estimate the gallic acid in the culture filtrate. Reagents: Methanolic rhodanine solution (0.667% w/v): Prepared by dissolving 0.667 g of rhodanine in 100 ml of methanol.Potassium hydroxide (0.5 N): 2.8 gpotassium hydroxide dissolved in100 ml of distilled water.

The procedure of Hagerman and Butler (1978) was used to estimate the tannin content in different tannin sources. Reagents: Bovine serum albumin (BSA) 1.0 mg/ml: 10.0 mg of bovine serum albumin was dissolved in 10.0 ml of 0.2 M acetate buffer, pH 5.0, containing 0.17 M sodium chloride. Sodium dodecyl sulfate (SDS)-triethanolamine solution: The solution contained 1.0% SDS and 5.0% (v/v) triethanolamine in distilled water. Ferric chloride reagent (0.01 M): 1.62 g of ferric chloride was dissolved in 1.0 L of 0.01 N hydrochloric acid.

The total protein content in the culture filtrate was estimated by Lowry’s method as described below: Reagents: Solution A: 2.0% Na2CO3 in 0.1 N NaOHSolution B: 0.5 % CuSO4 in 1.0 % Sodium potassium tartarate Solution C: 50.0 ml of solution A was mixed with 1.0 ml of solution B Folin Ciocalteau’s reagent

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

For bacterial isolates, a single colony from a nutrient agar slant was inoculated into 50 ml of nutrient broth in a 250 ml Erlenmeyer flask. These flasks were incubated at 37±1°C in a incubator shaker till an optical density of 0.6 at 660nm. Now these cultures were used to inoculate 50 ml of the tannase production medium in 250 ml Erlenmeyer flasks using 2% v/v inoculum. These flasks were incubated at 37±1°C in an incubator shaker (Multitron AG-27; Switzerland) at 200 rpm for 72h. The experiments were carried out in triplicates. Samples (2.0 ml for bacteria and same for fungi) were withdrawn at regular intervals of 12h upto 72 h. The samples thus obtained were centrifuged at 10,000 rpm in a refrigerated centrifuge (SIGMA 4K15 Germany) for 10 min at 4°C. The supernatant/s were analyzed for tannase activity

For fungal cultures, spores were harvested from 72 hour old cultures grown on PDA/Tannic acid agar slants by adding 10 ml of sterilized normal saline and a few drops of sterilized Tween-80 followed by vortexing. The spore suspension was filtered through sterile cotton filter to ensure that mycelial filaments are removed. The spores were counted using a haemocytometer (Neubaeur). Approximately, 5X106 spores were inoculated in 50 ml of tannase production medium in 250 ml Erlenmeyer flasks. These flasks were then incubated at 30±1 and 37±1°C in an incubator shaker (model G25KC, New Brunswick Scientific, NJ, USA) at 200 rpm

Nutrient agar (NA) medium The composition per litre of the medium is as follows: Peptone : 5.00 g Sodium chloride : 8.00 g Beef extract : 1.50 g Yeast extract : 1.50 g Agar-agar : 20.0 g Double distilled water : to make the final volume 1000 ml (iii) Tannic acid agar (TAA) medium This medium was used for screening of tannase producers. The composition per litre of the medium is as follows: Tannic acid : 10.00 g Agar-agar : 30.00 g Citrate phosphate buffer : to make the final volume 1000 ml (0.1M, pH 5.0) 30.0 g of agar-agar was melted and subsequently autoclaved. Citrate phosphate buffer and 0.1% (w/v)tannic acid, filter sterilized through 0.22μmembrane filters, were added to the sterilized molten agar and the final volume was made 1.0 L. (IV) Czapek Dox minimal medium (modified for tannase production)The composition per litre of the medium is as follows: Ingredients Fungi Bacteria Tannic acid : 10.00 g 10.00 g D-Glucose : 10.00 g 0.50 g NaNO3 : 6.00 g – NH4Cl : – 1.0 g KH2PO4 : 1.52 g 0.50 g K2HPO4 : – 0.50 g KCl : 0.52 g – MgSO4.7H2O : 0.52 g 0.50 g CaCl2 : – 0.01 g Cu(NO3)2.3H2O : trace – FeSO4.7H2O : trace – ZnSO4.7H2O : trace – Double Distilled water : to make 1.0 L to make 1.0 L pH : 5.0±0.2 5.0±0.

Potato dextrose agar (PDA) medium The composition per litre of the medium is as follows: Ingredients g/l Peeled and sliced potatoes : 200.0 Dextrose : 20.0 Agar-agar : 20.0 Double Distilled water : to make the final volume 1000 ml pH was adjusted to 6.2 ± 0.2 using 1N NaOH / HCl

Medium Compositio

Buffer pH range Stock Solutions Volume of Stock A + Stock BCitrate Phosphate 3–5 A: 0.1M solution of citric acid B: 0.2M solution of Na2HPO4pH 3: 39.8 ml A + 10.2 ml B made up to 100 ml pH 4: 30.7 ml A + 19.3 ml B made up to 100 ml pH 5: 24.3 ml A + 25.7 ml B made up to 100 ml Phosphate 6–8 A: 0.2M solution of NaH2P04 B: 0.2M solution of Na2HPO4 pH 6: 87.7 ml A + 12.3 ml B made up to 200 ml pH 7: 39 ml A + 61 ml B made up to 200 ml pH 8: 5.3 ml A + 94.7 ml B made up to 200 ml Tris - HCI 9 A: 0.1M solution of (HOCH2)3CNH2B: 0.1M HCI solution (16.16 ml of 11.35N HCI /L) pH 9: 70 ml A + 30 ml B made up to 200 ml Glycine - NaOH 10 A: 0.1M glycine B: 0.1M NaOH solution pH 10: 50 ml A + 32 ml B made up to 200 ml Phosphate hydroxide 11 A: 0.05M Na2HP04B: 0.05M sodium hydroxide pH 11: 91 ml A + 9 ml B Hydroxide Chloride 12 A: 0.05M KCI solution B: 0.05M KOH solution pH 12: 82 ml A + 18 ml B

Composition of buffers

1N Hydrochloric acid (HCl)* One normal hydrochloric acid was prepared by adding 1.0 ml concentrated HCl to 10.0 ml of double distilled water. •1N Sodium hydroxide (NaOH)* One normal sodium hydroxide was prepared by dissolving 4.0 g of NaOH in 100 ml of double distilled water. *: These were used for adjusting the pH of the medium. •Tween-80Tween-80 used as surfactant was prepared by adding 100 μl of concentrated Tween-80 to 100 ml of double distilled water and autoclaved.

tock Solution

Pharmacia (Uppsala, Sweden).Column chromatography matrices (Sephadex G series), DEAE-Cellulose E-Merck Germany Silica Thin Layer Chromatography Plates 60 F254, All solvents used in the present investigation were purchased from E-Merck Sisco Research Laboratories (SRL), SD fine chemicals Ltd, Qualigens, Central Drug House (CDH), Thomas BakerChemicals/components used in the preparation of various media were obtained from these companies Local commercial sourcewheat bran, rice bran, wheat straw, corn cob, acacia arabicajambula leaves, aamla, Indian plum, jowari, black tea, kangra orthodox black tea Double distilled water (DDW) was used for preparation of reagents, stock solutions, buffers and different media. All glass and plastic wares used were from Borosil, Schott Duran and Qualigens.

Tannic acid, DEAE-cellulose, phenyl sepharose, acrylamide, bisacrylamide, TEMED, ampholine PAG plates, bromophenol blue, chitosan, chitin, silica, celite, DEAE-sephadex, amberlite XAD-7, glutaraldehyde, cholic acid, saponin, sodium taurocholeate, SDS, tauro cholic acid, sodium choleate, triton X-100, Tween-80, EDTA, phenyl methyl sulfonyl fluoride, p-Chloromercuric benzoic acid, N bromosuccinimide, Phenyl boronic acid, O-phenanthrolin, sodium deoxycholate, phenanthrolin, N-ethylmaleimide, dithiothreitol, β- Mercaptoethanol, bromoacetic acid. gallic acid and its esters (methyl, propyl, ethyl, butyl gallate), epigallocatechin gallate , epigallocatechin, caffeine, epicatechin, epicatechin gallate, 2,2-diphenyl-1-picrylhydrazyl (DPPH) (E)-2-hexenal (Z-3-hexenol), 1-penten-3-ol, 3,7-dimethyl-1,5,7-octatrien-3-ol, linalool, linalool oxides (furanoid), geraniol, methylsalicylate, epoxylinalol, α-irone (2,5-dimethyl-α-ionone),2,7-epoxymegastigma-4,8-diene and 1,3-dioxolane

Chemicals and Reagents

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

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

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

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

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

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

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

Chemicals

Antibiotics

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

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

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

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

Buffers and solutions

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

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

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

Media

Table 2.3: Oligonucleotide primers

Primers

Table 2.2 List of plasmids used in this study

Plasmids

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

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

m. +

no material difference

historical circumstances dctennine the characteristics and purposes of social institutions and individual actions

.

different countries and re-mote ages, wherein the speakers and writers had very different notions, tempers, customs, orna-ments, and figures of speech, &c., every one of which influenced the signification of their words

StrongDefenseofrhetoricposthumousl

a set of technologically inter-linkedmaterial culture

Know ye that by “the world” is meant your unawareness of Him Who is your Maker, and your absorption in aught else but Him. The “life to come,” on the other hand, signifieth the things that give you a safe approach to God, the All-Glorious, the Incomparable.

material forms.

Make a wet version of the material

We have introduced a novel approach to time management using a device embodying characteristics of both an ambient display and a tangible user interface.

material transfer agreement (MTA)

MTA

material transfer agreement

Prowniananalysis

"it's not about the technology" because "the technology is neutral."

Park-specific documents

Finally, and perhaps most importantly, online reading may require even greater amounts of higher-level thinking than offline reading. In a context in which anyone may publish anything, higher-level thinking skills such as critical evaluation of source material become especially important online.

face the flushed, feverish, quaky, shaky, angry, scared, hurt, shocked, disappointed, alarmed, outraged, even terrified conditio

have to start searching that person's history until we begin to understand what led him or her to speak just so. Sometimes we do less well: if the history isn't there for us, we don't learn it, but instead make it up to suit ourselves.

The first step in testing claims of conspiracy is to establish precisely what is being claimed

Part V. Connexion of Place

One of those values is the principle of material honesty. One material should not be used as a substitute for another. Otherwise the end result is deceptive.

the material and the formal

hackers forming groups and follow this path (el8, h0n0, Zero for 0wned, project m4yh3m, etc).

Years later, the lottery winners were interviewed again, this time the results were quite surprising. As it turns out, their happiness level had dropped significantly to levels so low that most of the winners where more unhappy now than before winning the lottery. In contrast, the happiness of the group of paraplegics was very high, equal to or higher than before the accident.

Creating simulations, however, requires expert-level skills in interaction design, graphics, database functionality, and programming - not to mention instructional design, content expertise, and imagination.

As a genius of construction man raises himself far above the bee in the following way: whereas the bee builds with wax that he gathers from nature, man builds with the far more delicate conceptual material which he first has to manufacture from himself. In this he is greatly to be admired, but not on account of his drive for truth or for pure knowledge of things.

Cicero, too, in one passage, calls the material of oratory the topics which are submitted to it for discussion, but supposes that particular topics only are submitted to it.