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Longer probes derived from LTR restriction fragments were analysed in similar experiments to search for potential binding factors. The DNA binding affinity of the protein was confirmed by competition with oligomers specific for polytropic sequences added to the binding reaction. To act as a positive control for the reaction conditions, a probe previously used to demonstrate the presence of the same negative control region in the MLV LTR as above (Flanagan et al. 1989) was tested. The frequency of restriction sites in the pMX33-b construct meant that it was necessary to isolate and gel-purify a Pstl/Bglll fragment before synthesising the probe. A 120 bp Pstl/Haelll fragment, shared between polytropic and modified polytropic proviruses, was subsequently cut from this region (see Figure 6.1), end-labelled and used in EMSA experiments. Gel retarded products were clearly seen using nuclear protein extract from both 129 and C57BL/6 mice, and the specificity of the binding was confirmed by competition with the R lf/r oligomer which contains the binding sequence of the negative control factor (data not shown).

To analyse the poly tropic-specific sequence covered by the oligonucleotides PL037 and PL038, a 95 bp Apal/Mscl fragment was isolated from the polytropic pMX40-b construct, de-phosphorylated and end-labelled (see Figure 6.1). Binding reactions were set up with

129 C57BLV6 129 C57BL/6 ■<--- ► -4--- ► -4---► -4--- ►

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Figure 6.2

Figu re 6.2: B in d in g f a c t o r s s p e c i f i c to o l i g o m e r P L 0 3 7 / 3 8 . The polytropic LTR Apal/M scI end-labelled probe was added to nuclear protein extract from the m ouse strains indicated. The final NaCl concentrations in the binding reactions were as follows: 5m M , lanes 2 and 6; lOmM, lanes 3 and 7; 25m M , lanes 4, 8, 10 to 13; 50m M lanes 5 and 9. For binding com petition, 50- (lanes 10 and 12) or 100-fold (lanes 11 and 13) excess of cold oligom er P L 037/38 were added. Gel retarded products are marked with arrows. Lane 1 contains probe with no added protein extract.

nuclear protein preparations purified from thymus tissue taken from 129 and C57BL/6 mice and an example is shown in Figure 6.2. Lane 1 contains only the labelled probe, showing the mobility without added protein extract. Larger bands visible in the additional lanes therefore correspond to gel-retarded products caused by protein binding, as demonstrated by the bands marked in lanes 2 to 9. The specificity of these four bands was determined by competition with cold double-stranded oligomer PL037/38 in lanes 10 to 13. Three bands were eliminated, representing specific binding of a nuclear protein extract to a sequence in the PL037/38 oligomer. However, the experiment also demonstrated that this factor is not a candidate for Gvl, as the same bands are present in protein purified from both 129 (lanes 2 to 5, 10 and 11) and C57BL/6 mice (lanes 6 to 9, 12 and 13).

The U3 region covered by the PL035/36 oligomer was examined in similar experiments using a 113 bp Ddel/Ddel probe, isolated from a Pst/Bglll U3 fragment (see Figure 6.1). Although two gel-retarded products were observed, neither showed any sign of competition with the PL035/36 oligomer, and no difference in intensity of the bands could be seen between the two strains of mice.

Finally, a probe was designed to examine the possible binding of factors to both polytropic target regions simultaneously. A 298 bp SacI/MscI fragment, cut from the pMX40-a construct, was digested with Bsrl to create a 130 bp probe (see Figure 6.1). The results of an EMSA using this probe can be seen in Figure 6.3. Of the four gel-retarded products observed (numbered 1 to 4), bands 1, 3 and 4 appeared to show a reduction in intensity with increasing amounts of PL035/36 oligomer present in the binding reaction (lanes 4 to 7). Two of these bands (1 and 3), could not be eliminated using the PL037/38 oligomer as competition, suggesting that a specific binding factor was responsible (lane 8). Once again, these factors are not candidates for Gvl, as identical patterns of bands were observed from 129 (lane 2) and C57BL/6 (lane 3) mice. Band number 4 is therefore derived from a non­ specific association. Finally, no change in intensity of the fourth gel-retarded product

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F ig u re 6.3: B in d in g factors s p e c if i c to o l i g o m e r P L 0 3 5 / 3 6 . T he polytropic LTR BsrI/MscI end-labelled probe was added to nuclear protein extract from the m ouse strains indicated. F or binding com petition, 10-, 25-, 50- and 100-fold (lanes 4 to 7, respectively) excess o f cold oligom er P L 0 3 5 /3 6 was added. Lane 8 contains a 100-fold excess of cold oligom er P L 0 3 7 /3 8 and lane 1 contains probe with no added protein extract. Gel retarded products are marked with arrows.

(number 2) was observed, demonstrating the binding of a protein to a region of the probe not covered by the oligomers.

Although no G vl candidates emerged, these experiments did show some evidence of polytropic-specific binding factors in this region of the MLV LTR. However, these data were somewhat inconsistent, considering that the binding products that could be competed for by the PL035/36 oligomer were observed when using the BsrI/MscI but not the Ddel/Ddel probes. It was therefore decided to examine the U3 region by DNase footprinting for additional binding sites not covered by the oligomers tested above.