A nivel nacional

than with U extracts (Shephard et ah, 1994). This region (-6 to -38bp) was used in a gel retardation experiment to study further its involvem ent in the phénobarbital response. Oligonucleotides were synthesised corresponding to -38 to -13bp of the positive strand, and -30 to -6bp of the negative strand of

CYP2B2. The two oligonucleotides were annealed together and radiolabelled by fining in the ends as described in section 2.10.1a.

Figure 3.11 shows the gel retardation experiment with this fragment. N o complexes were formed with this fragment at all. This suggests that any binding of proteins to this region relies on stabilisation due to interactions with either another region of the promoter, or proteins bound to the promoter elsewhere.

P r o b e : - 6 t o - 3 8 b p C Y P 2 B 2

Treatment U PB PB PB —

SPl competitor — — -f- — —

Proteinase K — — — -1- —

T r a c k 1 2 3 4 5

Figure 3.11: Gel retardation with the -6 to -38bp fragment of the CYP2B2

promoter. The -6 to -38bp fragment was radiolabelled and incubated without protein (-), or with 5pg of nuclear protein extract made from the livers of untreated rats (U) or rats treated with phénobarbital (PB). Each reaction contained 2|ig of poly dl.dC. The reaction in track 3 included a 100-fold molar excess of the unlabelled -6 to-38bp fragment and the reaction in track 4 was incubated with 5pg proteinase K.

3 2 3 Gel retardation w ith the -34 to -86bp region of CYP2B2 gene promoter.

Although the presence of a C/EBP binding site in the -44 to -67bp region of

CYP2B2 had been demonstrated, this region did not show any differences in the abundance of complexes formed with PB extracts as compared to U extracts, so I was no further on in trying to clarify the PB induction mechanism of this gene.

The next approach was to return to the original result (Shephard et al, 1994), where the region -34 to -86bp did show a difference in the abundances of complexes formed with PB extracts as compared to U extracts. Scott et aL

(1994) observed that a shorter oligonucleotide (16bp) had a lower affinity for its binding protein than the corresponding 30bp oligonucleotide. It is possible that this is the case for the short -44 to -67bp oligonucleotide (23bp), so that the full effect is not seen. It was also possible that the changes I had made in extracting the proteins and carrying out the gel retardation experiment had altered this result. The -34 to -86bp fragment was generated by P.C.R. as in section 3.1.1, radiolabelled and used in a series of gel retardation experiments.

Several complexes were formed with both U and PB extracts (figure 3.12). Complexes in groups A and B all appear to be increased in abundance in response to phenobarbital-treatment of the rats, particularly complex 3. Over the series of experiments with this fragment there was a variation in the relative abundances of these complexes in group A and B, such that there did not appear to be a difference between the U and the PB extracts in some cases. This variation could be due to the different preparations of nuclear

P r o b e : -3 4 t o - 8 6 b p C Y P 2 B 2 Protein (jig) 2.5 5 7.5 5 — Treatm ent U PB U PB U PB PB — Proteinase K — — — — — — + — T r a c k 1 2 3 4 5 6 7 8 B

{

{

{

Figure 3.12: Gel retardation with the -34 to -86bp fragment of the CYP2B2 gene promoter. The -34 to -86bp region of the CYP2B2 gene was incubated without nuclear proteins (-), or with varying amounts of nuclear protein extracts made from the livers of untreated rats (U) or rats treated with phénobarbital (PB). The amount of poly dl.dC included in the reactions was 0.4x the amount of nuclear protein, or 2pg in the absence of nuclear protein. The reaction in track 7 was L 5|ig of proteinase K. The different protein-DNA complexes are grouped by letter to the left of the photograph.

mcubated wit numbered anc

proteins and different preparations of purified P.C.R. product used during the course of these experiments. It is also probable that the ratio of unlabelled to labelled -34 to -86bp DNA fragment would vary with each labelling reaction. The unlabelled DNA would act as a competitor, possibly masking effects seen w hen there was less unlabelled DNA present in the

j#*

reactions. However,^ in every case complex 3 was 1.5- to 4.3-fold more abundant with the PB extract than with the U extract. It was also observed that complex 6 was only present in reactions carried out in the presence of PB nuclear protein extract.

There were two complexes formed in group C, the lowest molecular weight one is more abundant with the U extract than the PB extract. Again there was a variation between 1.5- to 5-fold more of complex 1 with the U extract than with the PB extract over the series of experiments. It should be noted that not all of the relative abundances of the complexes varied in this way. The larger of the two complexes in group C was between 1.25- to 1.5-fold more of the complex with the U extract than with the PB extract throughout the series of experiments.

The difference in abundance of complexes formed with 2.5pg of nuclear protein extract as compared to 5jig of nuclear protein extract was m uch greater than the difference between 7.5pg and 5pg of nuclear protein extract. In the case of the PB extract, this could be due to the probe becoming limiting with 7.5pg nuclear protein (there is very little free probe), so not all of the possible complexes are seen. However, with the U extract where the same large increase in abundance of complexes between 5pg and 7.5pg was seen, the probe is still in excess with 7.5pg nuclear protein. Also, the complex with the slowest mobility was not seen until 5pg of nuclear protein extract was added to the reaction. These results suggest some extra

- 1 4 1 -

stabilisation of the protein-DNA complexes at a higher protein concentration.

This gel retardation (figure 3.12) w as compared to one using the -44 to -67bp fragment of the CYP2B2 gene promoter (figure 3.9). The complexes formed with the -44 to -67bp fragment were the same as those formed in groups B and C with the untreated extracts. So increasing the length of the gel retardation probe from -44/-67bp to -34/-86bp resulted in the formation of several extra higher molecular weight complexes (group A), and a difference between the abundances of complexes formed with the U and PB extracts.

A supershift gel retardation assay was carried out using the -34 to -86bp probe and anti-sera to C/EBPa, C/EBPp and C/EBP5 to determine the involvement of these forms in the complexes (figure 3.13). The complexes are numbered to the left of the photograph. Complex 1 forms in greatest abundance with U extracts and complex 3 is the complex predominantly increased in response to phénobarbital treatment of the animal. In this experiment complexes 4 and 5 also increase with the PB extracts.

Complex 5 is supershifted when anti-C/EBPa is included in the gel