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J. Biol. Chem., Vol. 275, Issue 51, 40218-40225, December 22, 2000

This Article Full Text Full Text (PDF) All Versions of this Article: 275/51/40218    most recent M909658199v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Becker, N. A. Articles by Maher, L. J. Search for Related Content PubMed PubMed Citation Articles by Becker, N. A. Articles by Maher, L. J., III Social Bookmarking                      What's this?

In Vitro and in Vivo Ligation-mediated Polymerase Chain Reaction Analysis of a Polypurine/Polypyrimidine Sequence Upstream of the Mouse metallothionein-I Gene^*

Nicole A. Becker, Heather A. O'Neill, Jeff M. Zimmerman, and L. James Maher III

From the Department of Biochemistry and Molecular Biology, Mayo Foundation, Rochester, Minnesota 55905

The mouse metallothionein-I homopurine/homopyrimidine (MT-I R/Y) sequence is a 128-base pair element located ~1.2 kilobase pairs upstream of the MT-I gene. Previous in vitro studies 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 vitro and in vivo analysis of the MT-I R/Y sequence using chemical probes of DNA structure and ligation-mediated polymerase chain reaction. In vivo analysis suggests neither profound base unpairing nor protein binding within the MT-I R/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 vitro does not imply the occurrence of such a structure in vivo. We were able to show both in purified genomic DNA and in vivo that 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 KMnO₄ reactivity 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 KMnO₄ modification of thymines.

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