Figure 1.

Xenopus laevis Oocyte Proteins Bind Specifically to the Mouse oct4 Promoter

(A) Diagram of the 2.3 kb regulatory region of the mouse oct4 gene (not to scale) [30]. The region contains a distal enhancer (DE), a proximal enhancer (PE), and a promoter (P). Translation is initiated at nucleotide +1. Four methyl-sensitive restriction sites are shown (M). Double-stranded DNA oligo fragments were designed to represent fragments of the oct4 regulatory region, as aligned below the diagram.

(B) oct4 promoter fragments (oligos) and their corresponding DNA sequences.

(C) Specificity of protein binding to oligos was determined by competition experiments, as shown here for the −166 oligo. Oocyte extract was added to P³²-labeled oligos in the presence of an increasing amount (12.5–50 μM) of competitor. The addition of the same but unlabeled oligo resulted in a stronger decrease of the gel-shift signal than the addition of an unspecific oligo of the same length that is not represented in the oct4 upstream region. This illustrates specificity for protein-oligo complex formation, which was found in all oligos.

(D) Xenopus oocyte proteins are competed out less efficiently with methylated oligos compared to the same oligos that are unmethylated. Competition with increasing concentrations (12.5–50 μM) of unlabeled oligos (unmethylated or methylated) revealed a protein binding preference for unmethylated oligos (only shown for the −166 oligo). The following abbreviation is used: methylated (Met).