Chemical synthesis of a mitochondrial gene designed for expression in the yeast nucleus

Abstract

A novel DNA sequence coding for subunit 8 of the mitochondrial ATPase of Saccharomyces cerevisiae has been constructed by chemical synthesis. The synthetic gene, termed NAP1, is designed for expression in the yeast nucleus and codes for a 48 amino acid polypeptide identical to that encoded by the mitochondrial aap1 gene of S. cerevisiae. The codons chosen for the NAP1 sequence correspond almost exclusively to those most frequently occurring in highly expressed yeast genes. The NAP1 coding region differs in 31 codons from that of aap1, and is flanked by sequences carrying restriction enzyme sites useful for cloning and for gene expression. A 170 bp double stranded DNA molecule was constructed by assembling 12 oligonucleotides (12 to 45 bases in length) in a single annealing/ligation mixture. This synthetic gene will provide a route for the systematic manipulation, through in vitro mutagenesis, of the structure of a protein normally encoded by mitochondrial DNA.