Page 1 \ ?^p 6r.1 CELL CYCLE CONTROL OF HUMAN H4 ...
Page 1 \ ?^p 6r.1 CELL CYCLE CONTROL OF HUMAN H4 ...
Page 1 \ ?^p 6r.1 CELL CYCLE CONTROL OF HUMAN H4 ...
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Aziz F: Cdl Ced. Cd*ol ol <strong>H4</strong> Gse ran&donan<br />
lo radio-lab€ll€d ONA iraqments ls €ssess6d by comp€ttion assays n which<br />
10 - 100lold €xc€ss ol unlabell6d oligonuceotides is added 10 lhe binding<br />
reaction. As posilive carnrols, oligonuclaotjdos wilh lhe same or slmilar<br />
sequ€nce ar€ added as sp€cfic comp€lrtor DNA, whereas unrelaled or mutanl<br />
DNA lragmenls arc vsed as negativ€ conlrors. To reduce non.specitic binding<br />
of prot€ins to probe DNA, bindifg feaciions also cornain a relatvely la€€<br />
amount ot non-sp€ciiic DNA. S6veral lyp€s ol non'specllic DNA subslrates<br />
can be us€d, inclldnq nalive oNA lrom salmon sperm ore cor, or synlhelic<br />
co polymeE such as poly (drdc)'poly (dl-dC)l'poly l/C oNA'l of poly(dc'<br />
dc)'poly(dG.dc)t'poi G/C ONA'I This lochniqu€ rs also uselullor idenlirying<br />
rhe actual prorein ihat binds to ths DNA; for example, anlibodies raised<br />
again$ proleins whch are belleved to bind to the DNA tragment, can be<br />
inc uded irilh€ r€action and lh6 6ft6cl oftofmarlon of prolein/DNA complexes<br />
In lhis sludy, gel shift assays w€ro used lo compare binding ol HiNF D,<br />
HiNF-M and H|NF-P in nuclear prcrein p.eparations lrom HeLa 53 cells to ONA<br />
tragm€nts spannlng wildtype and mL'lant B4-Site ll sequences (see Fig. 13)-<br />
Thelirsl sl€p in these qe shifr assays was to mak€ lhe probe whch spa.slhe<br />
Hind lrllMun Itragment ofth6 wildrype pFP-1 construct or analogous mutanr<br />
promoter consrrucrs. Plasmid oNAs wsre cut wirh Hind lll by incubalion in<br />
digestionbufi€l (50 /lot plasmid DNA(1&g/1rl),10 plo{ 10x NEB Buli€r+2,<br />
5 ,!l {20 units/rl) ot Hind rrr and 35 /rl oI Hro) ior several hou.s ar 37'c-<br />
Enzymes wer€ heat.inadivated at 65.C for 20 minut€s followed by elhanol<br />
59
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