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Using the FP5 oligonucleotide (Figure 3.11 A) as a probe, one major and one minor band with identical binding specificities were observed (Figure 3.1 IB ). These bands were efficiently competed for by the homologous, unlabelled FP5 oligonucleotide but not

Sequence of FP5 probe and competitors with the location and extent of the footprint within the tyrosinase promoter indicated. Lower case denotes residues not derived from the tyrosinase sequence while asterisks indicate mutated bases. The FP3 competitor was as described in Figure 3.9A while the EIIL competitor contains an SP1-binding site from the adenovirus Eli-Late promoter. B. Band shift using melanoma nuclear extract and FP5 probe competed with 20, 100 and 500-fold molar excess of the indicated homologous or mutant competitors. Identical results were obtained using HeLa cell nuclear extract (not shown). C. Competition for binding to the FPS probe by an oligonucleotide containing an SP1-binding site from the adenovirus EIIL promoter. D. The SP1 site Is conserved in evolution. Oligonucleotides spanning sequences located within the mouse, quail and turtle tyrosinase promoters from positions similar to that occupied by FPS in the human promoter were used as competitors at 20, 100 and SOO- fold molar excess.

. 1 FpS c t a g a T G A T G C g t t c G G T G G G A GTGGTATTATt F p S .M I * * * * * c t a g a T G A T G C T G G A G a a t t c A G T G G T A T T A T t F p 5 .M 2 c t a q a T G A T G C T G G A G G T G G G A cTcaTATTATt F p 5 .M 3 c t a g a T C T C GTGG GC GTGGTA G T C t E ll-L

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Probes and competitors: c t a a a T G A T G C T G G A G G T G G G A G T G G T ATTATt FP5 human C t a q a T G A T G T A A G A A G G G G A G T G G T TAt. FP5 mouse C t a a a C C T G T A G G G GG T G G GA AA AA AAGTCAGt FP5 quall c t a g a T G C T A A A G G T G C A G G A G T A A A A A A G T C A C t FP5 turtle Probe: Fp5 human oO”

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by a mutant, FP5.M 2, containing a four base substitution in the centre of the footprinted region. Mutation of residues 5' (FP5.M1) or 3' (FP5.M 3) from the centre of the FP5 footprint failed to inhibit binding by the FP5 factor. Consistent with the results obtained using the Fp3 probe, the factor recognising the FP5 oligonucleotide was not competed for using the FP3 oligonucleotide. Examination of the sequence bound by the FP5 factor revealed some similarities to a non-consensus SP1 motif, GTGGGCGTGGTAGT, found at a similar position in the adenovirus Ell-late (Ell-L) promoter (Goding et al., 1987). Using an oligonucleotide corresponding to the Ell-L SP1 motif as a competitor, the factor binding to the FP5 motif was competed for as efficiently as when using the homologous FP5 DNA (Figure 3 .110). This was confirmed using antisera raised against either recombinant of purified SP1, which, consistent with the results of others (Chen and Okayama, 1987), inhibited but did not completely abolish complex formation. These data indicate that the factor recognising the FP5 site in the tyrosinase promoter is SP1. Although the GGTGGGAGTGGT sequence recognised by SP1 in the human promoter is not completely conserved in other species, sequences present in the same relative position in mouse (AGGGGAGTGGT), quail (AGGGGGTGGGA) and turtle (AGGTGCAGGAGT) may also be able to bind SP1. To test this possibility, oligonucleotides spanning the putative SP1 recognition sequence from the mouse, quail and turtle promoters were used as competitors in a band shift assay together with the human FP5 probe and extract from B16 melanoma cells. The sequences of the competitors used and the results obtained are shown in Figure 3.1 ID . The competitors derived from mouse and quail were both able to compete for SP1 binding to the human FP5 probe as efficiently as the homologous DNA. By contrast no competition was observed using the DNA derived from the turtle promoter. Thus, although the primary DNA sequence is not entirely conserved, the human, mouse and quail promoters all bind SP1 at the same position relative to the M- box and CR2. To verify that this element was important for tyrosinase expression, the FP5.M2 mutation was introduced into the full length promoter extending to -300 and the level of CAT activity determined after transfection into B16 melanoma cells. The results (not shown) showed that mutation of the SP1 site resulted in up to an 8-fold decrease in promoter activity, consistent with a role for SP1 in tyrosinase expression.