Fig. 1. Binding of HeLa cell transcription factor TFIID to icefish myoglobin (Mb) promoter sequences. (A) Oligonucleotides corresponding to both strands of the Chaenocephalus aceratus upstream promoter sequence (-715 to -674) that contains the TATAAA duplication (in bold) (C. aceratus upstream promoter oligo), the homologous region (-666 to -640) of the C. rastrospinosus promoter (C. rastrospinosus upstream promoter oligo) and the conserved promoter sequence (-45 to -4) containing the putative `TATAAAA element' located within the core promoter region of both icefish (conserved TATAAAA element oligo) were synthesized based on the upstream promoter sequences obtained as described in Small et al., 1998. Numeric positions of sequences are relative to the transcription start site of each species. The TATAAAA sequence for both the C. aceratus upstream oligo and the conserved TATAAAA element oligo are underlined. Prior to use in the gel mobility shift assays, the complementary oligonucleotides were annealed and ³²P-end labeled using T₄ polynucleotide kinase and [-³²P]ATP prepared as described in the Materials and methods. (B) 2 pmol of the radiolabeled conserved TATAAAA element oligo (³²P-TATAAAA, lanes 1-5), radiolabeled C. aceratus upstream promoter oligo (³²P-Ace, lanes 6-10) or radiolabeled C. rastrospinosus upstream oligo (³²P-Ras, lanes 11 and 12) were incubated with (+) or without (-) 20 ng of purified human TFIID as indicated. Some samples also included 200 pmol of unlabeled conserved TATAAAA oligo (TATAAAA, lanes 3 and 7), C. aceratus upstream promoter oligo (Ace, lanes 4 and 8) or C. rastrospinosus upstream oligo (Ras, lanes 5 and 9) as competitor oligonucleotides. The DNA/TFIID complexes were then resolved by electrophoresis and autoradiography as described under Materials and methods. Arrows indicate the positions of shifted bands that presumably represent radiolabeled DNA/TFIID complexes.

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