Mitochondrial DNA polymorphism: evidence that variants detected by restriction enzymes differ in nucleotide sequence rather than in methylation.

Abstract

Restriction enzyme analysis of mt[mitochondrial]DNA for the purpose of determining sequence divergence rests on the assumption that variant recognition sites differ with respect to sequence and not methylation. This assumption was tested on 2 mtDNA, A and B, which are distributed throughout the laboratory rat population and can be distinguished by a number of restriction enzymes. The mtDNA were cloned and the nucleotide sequences of corresponding small HindIII fragments, in which a variant EcoRI site occurs, were determined. The fragments differed in sequence and not methylation. The cloned mtDNA yielded the same fragment pattern as did native mtDNA when treated with EcoRI, HhaI, HinfI, and HaeIII. Three nucleotide replacements were found in the 169 base pair fragment, A.cntdot.T .tautm. G.cntdot.C, A.cntdot.T .tautm. G.cntdot.C, T.cntdot.A .tautm. G.cntdot.C. One replacement, A.cntdot.T .tautm. G.cntdot.C at position 80, accounts for the presence of the EcoRI site in the type A and its absence in the type B mtDNA. Apparently the 3 nucleotide replacements are silent; i.e., they would not lead to amino acid substitutions in a possible encoded protein.