ALGUNOS ASPECTOS DE LA DISTRIBUCIÓN DE PRODUCTOS EN LA CADENA.

Either lO-lOOng of purified NFI or nuclear extracts containing 4pg (as determined by Bradford, 1976) of total protein were incubated with 20jxg BSA and Ipg of poly(dA.dT)poly(dG.dC) in 20pl of 150mM NaCl, 25mM Hepes-NaOH pH8,5mM MgCh, 20mM DTT and 0.01%

NP40. After 15 min at 20^C ^^P end labelled double stranded

oligonucleotide (l-2ng) containing the NFI DNA binding site was added and incubated at 20^0 for a further 15 min. For competition

experiments the assay mixtures were incubated with an excess of unlabelled double stranded oligonucleotides containing the NFI, NFlil or NF-kB DNA binding sites for 15min at 20^0 prior to the addition of 32P labelled double stranded oligonucleotides. 3]xl of dye (50%

glycerol, 150mM NaCl, 25mM Hepes, 5mM MgCh, 20mM DTT, 0.01% NP40 and 0.05% bromophenol blue) was added to each assay mixture prior to loading onto a 6% polyacrylamide gel (55:1,

acrylamide:N-N'-methylene-bis-acrylamide) and 50mM Tris borate, 0.5mM EDTA, pH8.3 which was polymerised by addition of 0.6% (v/v) of 25% APS and 0.06% (v/v) of TEMED (Kodak). Gels were run at 200 volts for 45-60 min in 50mM Tris borate, 0.5mM EDTA, pH8.3, fixed in

10% acetic acid for 10 min, dried and exposed to X-ray film at -TO^C with an intensifying screen. Alternatively each NFI-DNA complex was cut out of the dried gel, submerged in 15ml of scintillation solution (Ecoscint A, National Diagnostics) and its radioactivity determined by a SL 30 Liquid Scintillation Spectrometer (Intertechniques).

11. Purification of NFI from S.frugiperda cells Infected with recombinant baculovims

One litre of S.frugiperda cells were infected at 2pfu/ceU with recombinant baculovims containing either NFIfl or NFId b d cDNA (Bosher et al., 1991). Infected cells were incubated for 72 hours at 280C and were then collected by centrifugation. The cells were washed once and resuspended in 5ml of a lysis buffer containing 25mM Hepes-NaOH (pH8.0), ImM ethylene-diamine tetra-acetic acid sodium salt (EDTA), 2mM DTT, 0.4M NaCl, ImM benzamidine, ImM PMSF, ImM SMBS,

0.5% NP40 and lOjig/ml antipain, pepstatin and leupeptin. This was left on ice for 10 min and the cells were further dismpted with 10 strokes in a Dounce homogeniser using a type B pestle. After a further 20 min on ice it was centrifuged at 45000g for 30 min at 40C. The supematent was diluted 1 in 2 with column buffer (25mM Hepes-NaOH (pH8.0), ImM DTT, 10% (v/v) glycerol, 2mM SMBS, ImM PMSF and lOpg/ml antipain, pepstatin and leupeptin) and clarified by centrifugation at 45(XX)g for 15 min at 40C. To purify the NFI the clarified extract was applied directly to a column of Bio-Rex (Bio-Rad) equilibrated in

column buffer containing 0.2M NaCl. Unbound proteins were removed by washing the column with two bed volumes of column buffer

containing 0.2M NaCl. Bound proteins were eluted using 1ml aliquots of column buffer containing an increasing concentration of NaCl from 0.25M to l.OM (increasing in 0.05M steps). The fractions were

collected and their protein concentration determined by the method of Bradford (1976). The NFI DNA binding activity of these fractions was ascertained by gel electrophoresis DNA binding assays and scintillation counting of the NFI-DNA complex formed.

The fractions found to contain active NFI were pooled and diluted with column buffer to bring the NaCl concentration down to 0.25M. 6|Lig of poly(dl-dC) was added per mg of protein and then it was applied to a pre-equilibrated column containing the immobilised NFI-binding site linked via a 5'-amino link (Applied Biosystems) to CNBr-activated Sepharose (Pharmacia) as described (Clark et aL, 1990). Unbound proteins were removed by washing the column extensively with column volumes of column buffer. The bound NFI was eluted from the column in the same way as for the Bio-Rex column and the fractions tested for the presence of NFI as before.

12. Radiolabelling of DNA fragments

A plasmid pHRI, obtained from R. T. Hay, (Hay, 1985) that contains the 103bp Ad2 ITR was cut with EcoRI and Pst I to excise the Ad2 ITR which was then ^^P end labelled using the Klenow fragment of

E.coli DNA polymerase (Amersham). Ijig of the cut plasmid was incubated with 20pCi[a-^^P]dATP and unlabelled dGTP, dCTP and dTTP to final concentrations of lOOjiM in a buffer containing 50mM Tris (pH7.8), 5mM MgCh, ImM DTT and 100|ig/ml (w/v) bovine serum albumin (BSA) with 10 units of Klenow for 15 min at 20^0. Then unlabelled dATP was added to a final concentration of lOOjiM for a further 15 min at 20^0. The reaction was stopped by addition of 1/5 final volume of 50% glycerol, lOOmM EDTA and 0.05% bromophenol blue. Unincorporated dNTPS were separated from the labelled fragment by gel electrophoresis in an 8% polyacrylamide gel (29:1, acrylamide:N- N'-methylene-bis-acrylamide) containing lOOmM Tris borate, ImM EDTA, p H8.3 and was polmerised by addition of 0.6% (v/v) of 25% APS and 0.06% (v/v) of TEMED (Kodak). Gels were run at 200 volts for 45-60 min in lOOmM Tris borate, ImM EDTA, pH8.3. The band containing the labelled DNA fragment was located by autoradiography, excised from the gel and the DNA electroeluted.

13. DNase I footprinting

Labelled DNA (1.5ng) was incubated with varying amounts of

NFIf l, NFId b d and DBP in a final volume of 50jil containing 25mM Hepes-NaOH pH8.0, 5mM MgCb, 20mM DTT, 150mM NaCl, 0.01% N P40,0.5pg BSA and lOOng poly(dA.dT) and poly(dG.dC) for 60 min at 20^C. 0.25 units of DNase I (Amersham) was added and the

incubation allowed to proceed for 60 seconds at 20^C and the reaction stopped by the addition of 200|xl of 0.3M NaOAc, 20mM EDTA and

lOOfXg/ml tRNA. DNA was isolated by organic extraction followed by ethanol precipitation and resuspended in deionised formamide

containing 2mM EDTA, 20mM NaOH, 0.05% bromophenol blue and 0.05% xylene cyanole. The samples were mn on an 8% polyaciylamide gel (20:1, acrylamide :N-N'-methylene-bis-acrylamide) and containing

lOOmM Tris borate,ImM EDTA, pH8.3,46% urea and polymerised by addition of 1/200 volume of 25% APS and TEMED. Gels were mn in l(X)mM Tris borate, ImM EDTA, pH8.3 at 2000 volts at 50®C until the leading dye front was approximately 1 inch from the bottom. The gels were dried and exposed to X-ray film at -70^0 in the presence of an intesifying screen.