Expression of cloned mitochondrial DNA from the petite negative yeast Schizosaccharomyces pombe in E. coli minicells.

Abstract

The minicell producing Escherichia coli strain D24 (lysogenic for phage lambda cI857) was transformed with the recombinant plasmid pDG3 containing the entire mitochondrial (mt) genome of the fission yeast Schizosaccharomyces pombe (S. pombe) cloned in the single BamHI-site of the E. coli plasmid pBR322 (Del Giudice 1981). By DNA-RNA hybridization it could be shown that the total mtDNA sequence of the plasmid pDG3 was transcribed in the E. coli minicells. The cloned mtDNA also directed the synthesis of at least five novel polypeptides with molecular weights between 7,200 and 34,000. When the minicell producing E. coli strain P678-54 was transformed with the hybrid plasmid pDG3, considerable portions of the inserted mtDNA sequences were deleted. One of the resulting plasmids (pDG4), lacking about two-thirds of the mtDNA sequence, directed the synthesis of new polypeptides in the range of 7,000 to 17,500 daltons. Another derivative of pDG3, the plasmid pDG5, containing one-sixth of the mtDNA sequence, directed the synthesis of at least three novel polypeptides. The mt origin of novel polypeptides coded by the hybrid plasmid pDG3 was demonstrated by use of antisera raised against total mitochondrial proteins from S. pombe and antisera against subunits II and III of cytochrome c oxidase from Saccharomyces cerevisiae (S. cer.).