Manipulating the metazoan mitochondrial genome with targeted restriction enzymes.
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.
Xu
Hong
H
Department of Biochemistry and Biophysics, University of California at San Francisco, San Francisco, CA 94158-2200, USA.
DeLuca
Steven Z
SZ
O'Farrell
Patrick H
PH
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
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