Laboratory Reagents

2.1.1 Chemicals

General laboratory chemicals were of analytical grade and purchased from either Merck Ltd, Poole, Dorset, U.K.; Boehringer Mannheim, Lewes, East Sussex, U K ; Difco Laboratories, Detroit Laboratories, Detroit, U.S.A.; Sigma Chemical Company Ltd, Poole, Dorset, U K ; Life Technologies, Paisley, Renfrewshire, U K ; or Biometra Ltd, U .K Hexanucleotides [pd(N)6] for random prime labelling and dNTPs were obtained from Pharmacia Biotechnology Ltd, St. Albans, Herts, U .K Radiochemicals, Hybond™-N+ nylon membranes and Hybond™-C/Hybond™-C Extra nitrocellulose membranes were obtained from Amersham International Pic., Little Chalfont, Bucks, U.K.

Kodak X-OMAT imaging photographic film was purchased from Sigma Chemical Co. Ltd., Poole, Dorset, U .K , and Hyperfihn™-MP was obtained from Amersham Litemational Pic., Little Chalfont, Bucks, U .K Film developing and fixing chemicals were obtained from Photosol, Genetic Research Instrumentation. 30% (w/v) acrylamide/ 0.8% bisacrylamide stock solution for polyacrylamide gels was obtained from Scotlab.

General disposable plasticware was supphed by Greiner, except 2 0ml Universal Tubes and 10ml disposable pipettes which were supphed by Sterilin. 3MM chromatography paper was supphed by Whatman and disposable filters were obtained from Gelman Life

Sciences.

Ah sohd chemicals were dissolved in ddH2 0, adjusted to the required pH with HCl or NaOH and autoclaved or filter sterihsed unless otherwise stated. NaOH and SDS and SSC were not autoclaved; phenol and ethidium bromide solutions were stored in the dark. Chloroform always contained 4% isoamyl alcohol. Phenol (Fisher Scientific Company) was buffered by shaking three times with an equal volume o f 0.5M Tris (pHS.O), and then with 0. IM Tris (pHS.O) removing the aqueous layer each time.

2.1.2 General Solutions

(AU concentrations expressed at x l) Stock Concentration

PBS xlO 104mM sodium,

l.SmM potassium chloride,

5.4mM disodium orthophosphate dihydrate,

1.25mM potassium dihydrogen orthophosphate, pH7. Autoclaved and stored at room temperature.

SSC x20 150mM sodium chloride, 15mM sodium citrate, pH8. Stored at room temperature.

TE x l lOmM Tris-HCl pH7.4, ImM EDTA, pH8.0.

2.1.3 Enzymes

AU restriction Enzymes and DNA modifying enzymes were obtained from Promega, Southan^ton, U .K unless otherwise stated and stored at -20°C.

DNase-free RNase-A (Boehringer Mannheim, Lewes, East Sussex, U .K ) was prepared by dissolving lOmg ml'^ in 15mM NaCl, lOmM Tris-HCl, and boiling for 10 minutes to remove DNase activity. AUquots were stored at -20”C.

Table 2.1 - DNA Plasmids

P lasm id D escription R eferen ce/S o u rce pBS27 Chinese ham ster hsp27

cDNA in pBluescript SK+ (Stratagene Ltd., Cambridge, U.K.)

Dr. Jacq u es Landry Université Laval, Quebec, Canada.(Lavoie etal., 1990)

p59 pGem7Zf+ (Promega, Southampton, U.K.)

containing rabbit hsp56 cDNA

Dr. Marie-Claire Lebeau, Institut national de la sante et de la recherche médicale, Inserm U 33. (Lebeau etal., 1992)

pRep65 Human hsp60 cDNA in pRepS RSV-LTR driven expression vector (Invitrogen Corporation)

Prof. Radhay S. Gupta. (Jindal etal., 1989)

PH2.3 Human hsp70 cDNA in pAT 153 vector

American Type Culture Collection (Wu etal., 1985) ppa90 Human hsp90 cDNA cloned

into PHbA Pr-1-neo expression vector

Dept, of Molecular Pathology, University

College London (Twomey et al., 1993)

GM-BH pGem3Z vector (Promega, Southampton, U.K.)

containing Hinc\\/BamH\ deletion mutant of human HSF-1

Prof. Richard Voellmy, University of Miami, U.S.A. (Zuo etal., 1995)

pCH110 Eukaryotic a ssa y vector containing E. coli lacZ gene

Pharmacia Biotechnology Ltd, St. Albans, Herts, U.K pEGFPNI Eukaryotic expression vector

expressing green fluorescent protein (GFP) variant

optimised for expression in mammalian cells under the control of a CMV IE promoter, with an SV40 polyadenylation sequence

Clontech Laboratories Inc., Palo Alto,

California, U.S.A.

pUC-tub 800 bp fragment of human p- tubulin cDNA cloned into Pst\ site of pUCS plasmid vector

(Hall et ai, 1983)

pJ4Q Expression vector containing the Moloney murine leukaemia virus long terminal repeat (MoMLV- LTR) followed by a multiple cloning site and a 3' SV401 intervening sequence (IVS) and polyadenylation sequence

(Morgenstern and Land, 1990)

pJ7Q Expression vector containing the constitutive immediate early cytomegalovirus IE94 promoter (CMV IE94) with a 3' polylinker and a transcriptional termination signal

(Morgenstern and Land, 1990)

LSN HSP70 promoter driven CAT and neomycin resistance gene

plasmid

Dr. Richard 1. Morimoto Northwestern University, U.S.A.

(Williams and Morimoto, 1990)

pSP72 Ampicillin resistant E. coli cloning vector

Promega, Southampton, U.K.

pcDNAS Mammalian expression vector containing major CMV IE

prom oter/enhancer and 3’ bovine growth hormone (BGH)

polyadenylation sequence

Invitrogen Corporation

pNot3.5 3.5kb Noti fragment of RL region of herpes simplex virus type 1 (HSV-1) DNA cloned into pGem5Zf+ vector (Promega)

Dr. Robert S. Coffin, University College London

pDde rev 589bp Ddel fragment of HSV-1, including the LAT PI promoter, cloned into pGem3Z vector (Promega)

Dr. Robert S. Coffin, University College London

pA E IsplA pA E IsplB

Adenoviral shuttle vectors Dr. Lionel Wightman, United Medical and Dental Schools, U.K.

pCITE-1 Encephalomyocarditis virus internal ribosome entry site sequence in plasmid vector

Dr. Jeffrey Almond, University of Reading, U.K.

2 .2 Bacterial Strains and Growth Conditions

2.2.1 Bacterial Strains

D H 5a supE44 Alacl69 (0lacZAM 15) hsdRlV recAl

(Bethesda Research Labs, 1986) endAlgyrA96 thi-1 relAl

X Ll-BIue recAl endAl gyrA96 thi-1 hsdR17 supE44 relAl

(Stratagene Ltd., Cambridge U.K.) lac[F’proAB lacPZAMlS Tn70 (Tet)7

2.2.2 Propagation and Storage of Bacteria

Luria B ertani (LB) M edia: 1% (w/v) Bacto®-tryptone 1% (w/v) NaCl

0.5% (w/v) Bacto®-yeast extract

LB Agar:

LB containing 2% Bacto®-agar

Media were autoclaved at 120°C for 20 minutes at 10 lb square inch'\ Ampicillin (lOOmg ml'^ stock) [Sigma Chemical Corqpany, Poole, Dorset, U .K ] was stored at - 20°C. Tetracycline (5mg ml'^ in 50% (v/v) ethanol) [Sigma Chemical Coropany, Poole, Dorset, U.K.] was stored at -20°C.

Starter cultures oïE. coli were grown in 5ml o f LB containing no antibiotic, lOOfxg ml ^ o f anq)icillin or 15 tig ml^ o f tetracycline where required. Cultures were grown in a Gallenkamgp orbital shaker overnight at 37°C at 200rpm. lOOfil o f the overnight starter culture was used to inoculate 100ml LB for growth in preparation o f transformation- corqpetent cells. Larger volumes of LB to culture overnight for plasmid preparation were inoculated with 1 : 1 0 0 0 0 (v/v) of starter culture.

2.2.3 E. CO//Transformation

The method used for preparation and transformation o f competent cells is based on the calcium chloride method outlined in Sambrook et al., 1989.

The 100ml culture o f E. coli described in Section 2.2.2 was grown to an OD580 o f 0.4- 0.55 units and the bacteria were pelleted in 50 ml sterile tubes by centrifugation in a standard benchtop centrifuge at 3,000rpm at 4°C for 1 0 minutes. The supernatant was discarded and the tubes and lids wiped dry. The pellets were resuspended by shaking in 10 ml ice-cold lOOmM CaCL. The bacteria were again pelleted and drained as above, resuspended in 2ml/pellet of ice-cold lOOmM CaCL, and incubated on ice for at least 30 minutes prior to use. Conq)etent cells were stored on ice and used within 72 hours or discarded.

For transformation, 200p,l of conq)etent cell suspension was ahquotted into 15ml sterile tubes. The DNA for transformation was added to the cell suspension and mixed by flicking and incubated on ice for 30 minutes. The cells were heat shocked at 42°C for 90 seconds and cooled on ice for 2 minutes. SOOpl of LB medium was added and the cells were grown in an orbital shaker at 37°C/200rpm for 30 minutes to one hour. The cells were pelleted in a standard benchtop centrifiige at 22°C/3,000rpm, 900pl of supernatant was discarded, and the cells were resuspended in the remainder. The whole suspension was spread on selective LB plates supplemented with anq)icillin or tetracycline at 100|ig ml^ and 15pg ml'^ respectively. Plates required to demonstrate expression of (3-galactosidase were pre-treated with 50pl o f 4-Cl,5-bromo,3-mdolyl-y0-

galactosidase (X-gal, Insight Biotechnology Ltd) [20mg ml'^ stock in dimethyl formamide, stored in the dark at -20^*0]. Plates were incubated overnight at 37°C.