title: Further characterization of the respiratory deficient dum-1 mutation of Chlamydomonas reinhardtii and its use as a recipient for mitochondrial transformation.
abstract: The respiratory deficient dum-1 mutant of Chlamydomonas reinhardtii fails to grow in the dark because of a terminal 1.5 kb deletion in the linear 15.8 kb mitochondrial genome, which affects the apocytochrome b (CYB) gene. In contrast to the wild type where only mitochondrial genomes of monomer length are observed, the dum-1 genomes are present as a mixture of monomer and dimer length molecules. The mutant dimers appear to result from head-to-head fusions of two deleted molecules. Furthermore, mitochondrial genomes of dum-1 were also found to be unstable, with the extent of the deletion varying among single cell clones from the original mutant population. The dum-1 mutant also segregates, at a frequency of ca. 4% per generation, lethal minute colonies in which the original deletion now extends at least into the adjacent gene encoding subunit four of NAD dehydrogenase (ND4). We have used the dum-1 mutant as a recipient to demonstrate stable mitochondrial transformation in C. reinhardtii employing the biolistic method. After 4 to 8 weeks dark incubation, a total of 22 respiratory competent colonies were isolated from plates of dum-1 cells bombarded with C. reinhardtii mitochondrial DNA (frequency 7.3 x 10(-7)) and a single colony was isolated from plates bombarded with C. smithii mitochondrial DNA (frequency 0.8 x 10(-7)). No colonies were seen on control plates (frequency < 0.96 x 10(-9)). All transformants grew normally in the dark on acetate media; 22 transformants were homoplasmic for the wild-type mitochondrial genome typical of the C. reinhardtii donor. The single transformant obtained from the C. smithii donor had a recombinant mitochondrial genome containing the donor CYB gene and the diagnostic HpaI and XbaI restriction sites in the gene encoding subunit I of cytochrome oxidase (COI) from the C. reinhardtii recipient. The characteristic deletion fragments of the dum-1 recipient were not detected in any of the transformants.
authors.affiliation: Department of Botany, Duke University, Durham, NC 27706.
authors.forename: B L
authors.initials: BL
authors.lastname: Randolph-Anderson
authors.forename: J E
authors.initials: JE
authors.lastname: Boynton
authors.forename: N W
authors.initials: NW
authors.lastname: Gillham
authors.forename: E H
authors.initials: EH
authors.lastname: Harris
authors.forename: A M
authors.initials: AM
authors.lastname: Johnson
authors.forename: M P
authors.initials: MP
authors.lastname: Dorthu
authors.forename: R F
authors.initials: RF
authors.lastname: Matagne
mesh_terms: D000818:Animals; D001483:Base Sequence; D016825:Chlamydomonas reinhardtii / Q000235:genetics*; D004272:DNA, Mitochondrial / Q000235:genetics; D005111:Extrachromosomal Inheritance / Q000235:genetics*; D008040:Genetic Linkage; D008928:Mitochondria / Q000502:physiology*; D008969:Molecular Sequence Data; D009154:Mutation; D010101:Oxygen Consumption / Q000235:genetics*; D010641:Phenotype; D014170:Transformation, Genetic
chemical_list: D004272:DNA, Mitochondrial