Figure 2.

OsO₄ footprint of the (TG)₂₈ repeat of the rat ENK gene in the caudate nucleus and thalamus. (A) The solid arrowhead points to the most reactive thymidine at position −618 on the OsO₄ footprint performed on caudate nucleus (see also Fig. 1). The caudate nucleus (Cn) and thalamus (Th) were dissected from 40 adult rat brains and treated with OsO₄, and after the isolation of genomic DNA the 1,081-bp BglII fragment containing the endogenous (TG/AC)₂₈ repeat of the rat ENK gene (position −1,081, indicated by open arrowhead) was probed with a radioactively labeled fragment rENK505–592 (29). An identical segment of the ENK gene isolated from rat genomic DNA without OsO₄ treatment. (Lane B). Ten- and one-attomole standards (10, 1) derived from the 842-bp BglII–SphI fragment of the rENK gene (position −842 bp of the fragment is marked by arrowhead) illustrate detection sensitivity. Lanes T, C, G, and A show the sequencing ladders; the positions of the (TG)₂₈ and (TC)₂₀ repeat are marked. The sequencing primer overhangs the template segment by 8 bases; therefore, the sequencing ladder is shifted 8 bases above the genomic position. (B) Correlation between ENK mRNA levels and OsO₄ reactivity at the d(TG/AC)₂₈ repeat of the rENK gene in Cn and Th regions of the adult rat brain. Relative values were obtained from quantitive reverse transcription-coupled PCR and quantification of OsO₄ reactivity on a PhosphorImager.