GENERAL METHODS 2 1 1 Sterilisation

N on-sterile plasticware (SW41 tubes, eppendorf tubes etc.) was sterilised by

autoclaving at 121®C, 15 pounds/inch^ for 30 mins. Solutions were sterilised under the same conditions, or by filter sterilisation through a 0.45 /wm Nalgene disposable filter. For RNA work, no glassware was used, and all solutions were treated with DEPC (0.1% v /v . Sigma), prior to autoclaving. Protein purification columns were washed in 0.03% sodium azide (Sigma, NaNj) in 0.1 M column buffer (table 2.4) if they were to be redundant for more than 2 days.

2 .1 .2 . DNA extraction and precipitation

DNA solutions were treated with phenol chloroform to remove contaminating proteins. Phenol (BRL) was equilibrated with 10 mM Tris (BDH) adjusted to 7.5 mM with

1 M HCl (PSA laboratory supplies), and stored at -20®C or at +4®C in the dark. All chloroform (May and Baker) used was a 24:1 mixture of chloroform: isoamyl alcohol (Sigma). DNA solutions were extracted with an equal volume of phenol chloroform isoamyl alcohol mix (25:24:1), vortexed and m icrofuged at 13 K rpm in a Heraeus sepatech biofuge A for 5 min. The upper aqueous layer was retained, and re-extracted with chloroform , vortexed and microfuged again to remove phenol contaminants. The chloroform extraction step was repeated, before addition of 0.1 volume of sodium acetate (Sigma, pH 5.2), and 2 volumes of 96% ethanol (Hayman Ltd.) to the aqueous phase. Precipitation was perform ed either on solid COj for 20 min, or overnight at -20®C, and DNA collected by centrifugation for 25 min at 13K. The pellet was washed with 70% ethanol, air dried, and resuspended in

H^O.

2 .1 .3 . Determination o f nucleic acid and protein concentration

A Uvikon 860 spectrophotometer was used to determ ine the concentrations of nucleic acids in quartz cuvettes according to the formulae below (Sambrook et al., 1989)

1 A2(0 = 50 /ig/m l double-stranded DNA

1 Ajeo = 40 /ig/m l single stranded oligonucleotides 1 A2(0 = 40 /ig/ml RNA

1 A2(0 = 20 /ig/ml double-stranded oligonucleotides

Protein concentrations were determined by the use o f the Bio-Rad protein concentration assay, based upon the method of Bradford (1976). Protein solutions were added in a 4:1 ratio to the reagent, and absorbance measured at 595 nm in disposable cuvettes. The samples were quantitated with respect to a range of BSA (Sigma) standard concentrations.

2.1.4. Preparation of dialysis tubing

Dialysis tubing (Medical! international) was prepared by boiling in 10 mM EDTA (BDH, pH 8) for 2 hours prior to sterilisation by autoclaving, and stored at 4®C in 50% ethanol, 0.03% NaN,.

2.1.5. Autoradiography

Radioactive gels were autoradiographed by exposure to K odak X A R 5 film with an intensifying screen at -70®C unless otherwise specified. Films were developed by a F uji FMP 2100 x-ray automatic processor.

2.2. DNA USED

2.2.1. Synthetic Oligonucleotides

Synthetic oligonucleotides were prepared ’in-house’ on an Applied Biosystems automatic synthesizer. Oligonucleotides were deprotected by incubation in 35% ammonium solution (BDH) for 6 hours at 55®C, and ethanol precipitated. Complementary single­ stranded (ss) oligonucleotides were annealed by heating equimolar quantities to 90°C in ligase buffer (section 2.5.2), and cooling slowly to room temperature. The oligonucleotides used were :-

71/50:-w ild type binding sequence for D RTFl from E2A prom oter spanning -71 to -50 (La Thangue et al., 1990).

5 ' G ATC TAG TIT TCG CGC TTA AAT TIG A 3 ' 3 ' ATC AAA AGC GCG AAT TTA AAC TCT AG 5 '

64* :-as above, but with point mutation at position -64 (La Thangue et al., 1990).

5 ' G ATC TAG TTT TAG CGC TTA AAT TTG A 3 '

3 ' ATC AAA ATC GCG AAT TTA AAC TCT AG 5'

63* :-as above, but with point mutation at position -63 (La Thangue et al., 1990)

5 ' G ATC TAG TTT TCT CGC TTA AAT TTG A 3 '

3 ' ATC AAA AGA GCG AAT TTA AAC TCT AG 5 '

62* :-as above, but with point mutation at position -62.

5 ' G ATC TAG TTT TCG AGC TTA AAT TTG A 3 '

3 ' ATC AAA AGA TCG AAT TTA AAC TCT AG 5'

61* :-as above, but with point mutation at position -61.

5 ' G ATC TAG TTT TCG CTC TTA AAT TTG A 3 '

3 ' ATC AAA AGA GAG AAT TTA AAC TCT AG 5 '

60* :-as above, but with point mutation at position -60.

5 ' G ATC TAG TTT TCG CGA TTA AAT TTG A 3 '

3 ' ATC AAA AGA GCT AAT TTA AAC TCT AG 5 '

60/62:-as above, but with point mutation of positions -60 to -62 (La Thangue et al., 1990).

5 ' G ATC TAG TTT TCG ATA TTA AAT TTG A 3 '

3 ' ATC AAA AGA TAT AAT TTA AAC TCT AG 5 '

50/82:-sequence from -82 to -50 of the E2A promoter on the non-coding strand, annealed to -82 to -70 o f the coding strand (Shivji and La Thangue, 1991).

5 ' TGG AGA TGA CGT A 3 '

3 ' ACC TCT ACT ACA TCA AAA GCG CGA ATT TAA ACT 5 '

50/82 C R E -M -as above, except for 2 point mutations changing positions -72 and -73.

5 ' TGG AGA TGA TTT A 3 '

3 ' ACC TCT ACT AAA TCA AAA GCG CGA ATT TAA ACT 5 '

P :- Adenovirus E4 sequence from -58 to -39 (Gilardi and Perricaudet, 1984).

5 ' G ATC TAA CCG TTA CGT CAT TTT TT 3 '

3 ' ATT GGC AAT GCA GTA AAA AAC TAG 5'

PM l :-as above, but with point mutation of positions -48 and -49 (Tassios and La Thangue, 1990).

5 ' G ATC TAA CCG TTA AAT CAT TTT TT 3 '

3 ' ATT GGC AAT TTA GTA AAA AAC TAG 5 '

OCT :-Immunoglobulin heavy chain enhancer position -554 to -536 (Ephrussi et al., 1985).

5 ' G ATC CGG TAA TTT GCA TTT CTA A 3 '

3 ' GCC ATT AAA CGT AAA GAT TCT AG 5 '

O C T M l-as above, except that positions -546 and -547 are mutated.

5 ' G ATC CGG TAA TCG GCA TTT CTA A 3 '

3 ' GCC ATT AGC CGT AAA GAT TCT AG 5 '

S GLOBIN:-site C o ï p globin 3’ enhancer (Wall et al., 1988). K ind gift from E.DeBoer, NIMR.

5 ' CGT CAG GAT GTT TAA GAT TAG CAT TCA GGA AG 3 '

3 ' A GTC CTA CAA ATT CTA ATC GTA AGT CCT TCG C 5 ' GATA :-kind gift of S. Philipsen, NIMR.

5 ' TCG AGT ACT GCC TAT CTC AGC TCA A 3 '

Peptide 5 Antisense:-

5 ' CGC GGA TCC AAA TTT TGA CTC TCC CGA GC 3 '

GG C G GAT T G

C C

T T

Peptide 6 Sense:-

5 ' CGC GAA TCC CCA AAC ACA CAC TTC GT 3 '

G T G T T

C C

T T

2.2.2. Plasmid DNA

Plasmids used in this study are listed below. pE2Acat

This plasmid contains nucleotides -96 to +61 of the Adenovirus type 5 E2A transcription unit. The fragm ent was isolated by EcoKX D de\ restriction digestion.

pBIuescript II SK /KS"^

Prokaryotic cloning vector derived from PUC 19. A phagemid containing the FI phage ori and the colEl ori. Ampicillin resistant. Stratagene.

SP64y actin

Bam H l H indlll fragm ent from human 7 actin cDNA subcloned into pSP64 (Enoch et al., 1986). The plasmid was linearised with H in fl for SP6 polymerase reaction. Ampicillin resistant. K ind gift from E.Dzierzak, NIMR.

clone 6

p46 360 nucleotide cDNA cloned into the BarriHl site of pBIuescript II SK'. The plasmid was linearised with EcoK\ to give sense transcription with T3 RNA polymerase, and with Xba\ to transcribe antisense RNA from the T7 promoter. Ampicillin resistant.

myogenin

5’ end of myogenin cDNA, Sm al to P5/I, subcloned into vector GSMZ from S.-P. Yee, NIMR. 150 bp Myogenin fragm ent released with Sm al and Pstl.

EF-la

Xenopus E F -la Pstl Sacl fragm ent (Krieg et al., 1989), kind gift from R.Wilson, NIMR. 2.3. RESTRICTION DIGESTION OF PLASMID DNA

2.3.1. Diagnostic digestion

Diagnostic digests used 1-10 /ig DNA, and approxim ately 5 units (U) of enzyme//ig DNA, in the appropriate 1 X restriction buffer (table 2.1). Volumes of reactions were kept to a minimum, but the volume of enzyme never exceeded 10%. Reactions were perform ed at

37®C for 1 hour, DNA dye (table 2.2) was added to the reaction m ixture, and the products electrophoresed through a 0.7 - 1.4 % agarose (Bio Rad) Uniscience m ini-gel containing 0.5 /ig/m l ethidium bromide (Sigma) at 12V/cm for 1 hour, in 1 X TEE (table 2.2). M arkers (lam bda DNA digested with HindlW or HindlW and E coR l, or pBluescriptll KS^ digested with S d u \) were electrophoresed in parallel. Products were examined by ultra violet (uv) illum ination on a TMP 20 UVP transillum inator, and the gel photographed on Polaroid 667 film.

2.3.2. Preparative digestion

Preparative digestion of DNA was perform ed with upto 200 /ig DNA and 2 U enzyme//xg DNA in the appropriate buffer. Reactions were incubated for 3 hours at 37®C, and an aliquot assayed by electrophoresis as described above. Once digestion was complete, the sample was phenol chloroform extracted, ethanol precipitated, and the DNA taken up in 40 ijA dHjO. DNA dye was added, and the sample electrophoresed alongside size markers

in a 1% low melting point (LMP, Nusieve, GTG) agarose gel in 1 X TBE. The desired fragm ent was excised from the gel, and melted in an equal volume of TE (10 mM Tris-H C l (pH 8), 1 mM EDTA) at 65®C for 10 min. The molten sample was extracted twice in warm phenol chloroform , with vigorous vortexing. Once the interface was clear, the sample was chloroform extracted, and ethanol precipitated.