In Vitroand in VivoLigation-mediated Polymerase Chain Reaction Analysis of a Polypurine/Polypyrimidine Sequence Upstream of the Mousemetallothionein-IGene*

The mouse metallothionein-Ihomopurine/homopyrimidine (MT-IR/Y) sequence is a 128-base pair element located ∼1.2 kilobase pairs upstream of theMT-Igene. Previous in vitrostudies of this sequence in purified plasmids indicated the formation of a non-B DNA structure stabilized by acidic pH and negative supercoiling. We now present a detailed in vitroand in vivoanalysis of the MT-IR/Y sequence using chemical probes of DNA structure and ligation-mediated polymerase chain reaction. In vivoanalysis suggests neither profound base unpairing nor protein binding within the MT-IR/Y sequence before or after metal induction of MT-I. We conclude for this element that the propensity to adopt an unusual DNA structure in vitrodoes not imply the occurrence of such a structure in vivo. We were able to show both in purified genomic DNA andin vivothat only isolated thymines and the 3′ terminal thymine in strings of consecutive thymines are modified significantly by KMnO4, indicating an altered thymine accessibility pattern within the R/Y sequence. This KMnO4reactivity pattern is more consistent and predictable within the R/Y sequence when compared with flanking sequences. We propose a simple steric interference model to explain the observed pattern of KMnO4modification of thymines.