Amplification of a circular episome carrying an inverted repeat of the DFR1 locus and adjacent autonomously replicating sequence element of Saccharomyces cerevisiae

Abstract

Lack of suitable amplification markers has hindered the use of the yeast system for investigating the mechanism of gene amplification in a eukaryote with a simple genome and well defined genetic system. Recently, methotrexate has been used to select for Saccharomyces cerevisiae mutants with de novo amplification of the dihydrofolate reductase gene (DFR1) (Huang, T. (1993) In Vivo Disruption and de Novo Amplification of the DFR1 Gene Encoding Dihydrofolate Reductase in Saccharomyces cerevisiae. Ph. D. thesis, University of Alberta, Edmonton, Canada). We report here the detailed structure of a DFR1 episome amplified in methotrexate-resistant strain 25-1. The extrachromosomal DNA is found predominantly as a single 11-kilobase circular molecule. It consists of a 5.5-kilobase inverted duplication that contains the DFR1 locus and adjacent ARS (autonomously replicating sequence) element. This molecular configuration mimics the inferred structure of double minute chromosomes observed in a number of mammalian amplification systems and suggests that mechanisms that generate amplified DNAs are conserved from yeast to mammals.