Further characterization of the respiratory deficient dum-1 mutation of Chlamydomonas reinhardtii and its use as a recipient for mitochondrial transformation

Summary

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 × 10⁻⁷) and a single colony was isolated from plates bombarded with C. smithii mitochondrial DNA (frequency 0.8 × 10⁻⁷). No colonies were seen on control plates (frequency < 0.96 × 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.