In vitro and in vivo ligation-mediated polymerase chain reaction analysis of a polypurine/polypyrimidine sequence upstream of the mouse metallothionein-I gene.
The mouse metallothionein-I homopurine/homopyrimidine (MT-I R/Y) sequence is a 128-base pair element located approximately 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 KMnO(4), indicating an altered thymine accessibility pattern within the R/Y sequence. This KMnO(4) 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 KMnO(4) modification of thymines.
Becker
N A
NA
Department of Biochemistry and Molecular Biology, Mayo Foundation, Rochester, Minnesota 55905, USA.
O'Neill
H A
HA
Zimmerman
J M
JM
Maher
L J
LJ
D016475:3T3 Cells; D000818:Animals; D001483:Base Sequence; D004247:DNA; D008668:Metallothionein / Q000235:genetics*; D051379:Mice; D015335:Molecular Probes; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D011401:Promoter Regions, Genetic; D011687:Purines / Q000737:chemistry*; D011743:Pyrimidines / Q000737:chemistry*
D015335:Molecular Probes; D011687:Purines; D011743:Pyrimidines; D004247:DNA; D008668:Metallothionein