title: Manipulating the metazoan mitochondrial genome with targeted restriction enzymes.
abstract: High copy number and random segregation confound genetic analysis of the mitochondrial genome. We developed an efficient selection for heritable mitochondrial genome (mtDNA) mutations in Drosophila, thereby enhancing a metazoan model for study of mitochondrial genetics and mutations causing human mitochondrial disease. Targeting a restriction enzyme to mitochondria in the germline compromised fertility, but escaper progeny carried homoplasmic mtDNA mutations lacking the cleavage site. Among mutations eliminating a site in the cytochrome c oxidase gene, mt:CoI(A302T) was healthy, mt:CoI(R301L) was male sterile but otherwise healthy, and mt:CoI(R301S) exhibited a wide range of defects, including growth retardation, neurodegeneration, muscular atrophy, male sterility, and reduced life span. Thus, germline expression of mitochondrial restriction enzymes creates a powerful selection and has allowed direct isolation of mitochondrial mutants in a metazoan.
authors.affiliation: Department of Biochemistry and Biophysics, University of California at San Francisco, San Francisco, CA 94158-2200, USA.
authors.forename: Hong
authors.initials: H
authors.lastname: Xu
authors.forename: Steven Z
authors.initials: SZ
authors.lastname: DeLuca
authors.forename: Patrick H
authors.initials: PH
authors.lastname: O'Farrell
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D004262:DNA Restriction Enzymes / Q000235:genetics / Q000378:metabolism*; D004272:DNA, Mitochondrial / Q000235:genetics* / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific / Q000235:genetics / Q000378:metabolism*; D004331:Drosophila melanogaster / Q000196:embryology / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D004625:Embryo, Nonmammalian / Q000378:metabolism; D005123:Eye / Q000033:anatomy & histology / Q000254:growth & development; D005260:Female; D049750:Genome, Insect; D054629:Genome, Mitochondrial; D007248:Infertility, Male; D008297:Male; D028361:Mitochondrial Diseases / Q000235:genetics / Q000378:metabolism; D009024:Morphogenesis; D009132:Muscles / Q000648:ultrastructure; D009137:Muscular Dystrophy, Animal; D009154:Mutation; D013091:Spermatogenesis
chemical_list: D001426:Bacterial Proteins; D004272:DNA, Mitochondrial; D004262:DNA Restriction Enzymes; C024404:BglII endonuclease; C034510:CTCGAG-specific type II deoxyribonucleases; D015252:Deoxyribonucleases, Type II Site-Specific